Bioactive products of arginine in sepsis: tissue and plasma
composition after LPS and iNOS blockade
Mark J.
Lortie,
Shunji
Ishizuka,
Doron
Schwartz, and
Roland C.
Blantz
Division of Nephrology/Hypertension, University of California
San Diego School of Medicine and Veterans Affairs Health Care
System, San Diego, California 92161
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ABSTRACT |
Blockade or gene
deletion of inducible nitric oxide synthase (iNOS) fails to fully
abrogate all the sequelae leading to the high morbidity of septicemia.
An increase in substrate uptake may be necessary for the increased
production of nitric oxide (NO), but arginine is also a precursor for
other bioactive products. Herein, we demonstrate an increase in
alternate arginine products via arginine and ornithine decarboxylase in
rats given lipopolysaccharide (LPS). The expression of iNOS mRNA in
renal tissue was evident 60 but not 30 min post-LPS, yet a rapid
decrease in blood pressure was obtained within 30 min that was
completely inhibited by selective iNOS blockade. Plasma levels of
arginine and ornithine decreased by at least 30% within 60 min of LPS
administration, an effect not inhibited by the iNOS blocker
L-N6(1-iminoethyl)lysine
(L-NIL). Significant increases in plasma nitrates and
citrulline occurred only 3-4 h post-LPS, an effect blocked by
L-NIL pretreatment. The intracellular composition of organs
harvested 6 h post-LPS reflected tissue-specific profiles of arginine
and related metabolites. Tissue arginine concentration, normally an
order of magnitude higher than in plasma, did not decrease after LPS.
Pretreatment with L-NIL had a significant impact on the
disposition of tissue arginine that was organ specific. These data
demonstrate changes in arginine metabolism before and after de novo
iNOS activity. Selective blockade of iNOS did not prevent uptake and
can deregulate the production of other bioactive arginine metabolites.
L-N6(1-iminoethyl)lysine; agmatine; polyamines; arginine decarboxylase; ornithine decarboxylase; nitric
oxide; putrescine; ornithine; spermine; spermidine; lipopolysaccharide; inducible nitric oxide synthase
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INTRODUCTION |
THE CONCENTRATION OF ARGININE in extracellular fluid is
maintained within 100-200 µM mainly by 1) absorption
from the diet, 2) conversion to ornithine by the liver, and
3) synthesis from citrulline in the kidney (37). The importance
of an exogenous source of this "semi-essential" amino acid is
well known with respect to normal growth and wound healing. Thus,
although most cells can synthesize some arginine, the cellular uptake
route is thought to be of primary importance. A number of products
derived from arginine are known to be important bioactive compounds
affecting a broad range of physiological and pathophysiological
functions; however, regulation of the metabolic fate of this amino acid
is less well defined. In the last decade, an enormous amount of
interest has focused on the role of nitric oxide (NO) generated from
arginine by NO synthase (NOS). NO is a highly reactive vasodilatory
substance with other effects ranging from neuromodulation (47) to
bacteriostasis (50, 52). Arginine is also converted to ornithine, the
precursor to polyamines, via the action of arginase. The essential and
ubiquitous nature of polyamines (putrescine, spermine, and spermidine)
derived from ornithine decarboxylase (ODC) activity has been
characterized for decades (21, 22, 28), yet the mechanisms whereby
these cationic compounds regulate gene transcription and
Ca2+ signaling remain elusive. A regulatory role for
polyamines in inducible (i) NOS induction has been reported whereby ODC
activity is elevated before iNOS expression (33, 36), and the aldehyde metabolites of these compounds suppress iNOS induction (51). Evidence
of agmatine production by arginine decarboxylase (ADC) in mammals is
more recent, but there is increasing physiological and pharmacological
evidence of central, vascular, and renal actions of this compound (18,
24, 35). Because arginine is the common substrate for these molecules,
it is not surprising that feedback mechanisms for the different
metabolic pathways interact. For example,
N-omega-hydroxy-L-arginine, an intermediary in the
production of NO from arginine, is a potent inhibitor of arginase
activity (7), as is citrulline (44). Also, a report describes increased polyamine degradation mediated by exogenous agmatine (53). Work from
our laboratory has shown that agmatine exerts inhibitory effects on
both NOS and polyamine pathways (40, 43) and most recently that NO
directly inhibits ODC activity (39).
Sepsis is characterized by renal failure and a reduction in systemic
vascular resistance that is resistant to vasopressor therapy. Bacterial
products such as lipopolysaccharide (LPS) trigger a number of cellular
events via the immune response of cytokine release (6), including the
induction of a Ca2+-independent isoform of NOS (iNOS; see
Ref. 57) and massive NO production. Hypotension and impaired renal
function in the (LPS) rat model of sepsis is ameliorated by
administration of highly selective iNOS inhibitors (42). However, iNOS
blockade (1, 23, 49) or gene deletion (26, 54, 58) cannot fully
abrogate all the effects of LPS. This might be anticipated considering
the multiple adaptive mechanisms coinduced with iNOS. LPS is known to
upregulate synthesis of the Na+-independent Y+
transporters (9, 20), which facilitates the uptake of arginine and
ornithine. Increases in arginine transport may be necessary for the
generation of high NO levels (4, 48) and appears to be regulated
independently from de novo iNOS expression (11, 45, 48) and polyamine
synthesis (11). However, the net effect of upregulating bidirectional
transporters on intracellular amino acid concentration is not clearly
defined. In addition to iNOS expression, a multitude of cofactors and
urea cycle enzymes that may promote and/or regulate NO production are
upregulated by LPS (10, 17, 29, 32, 34, 46).
Considering the alternate pathways of arginine metabolism, the question
arises as to whether selective blockade of the iNOS enzyme, at a time
when arginine transport and/or synthesis is elevated, might promote the
production of other bioactive compounds derived from arginine. We
present herein a series of experiments in rats in which we assessed
LPS-induced changes in arginine disposition with respect to iNOS
induction in the presence or absence of
L-N6(1-iminoethyl)lysine
(L-NIL), a selective iNOS inhibitor. The data demonstrate
that multiple arginine metabolic pathways are upregulated in the LPS
model of sepsis. Furthermore, we show that early changes in plasma
amino acid composition precede de novo iNOS activity. We also establish
the impact of LPS with and without selective iNOS blockade on the
characteristic intracellular amino acid composition of the kidney,
lung, liver, and heart. Finally, we report a discordance in the
temporal expression of physiological responses to LPS with respect to
de novo iNOS expression, suggesting the occurrence of continuously
expressed iNOS.
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METHODS |
Animal preparation.
To determine temporal changes in plasma amino acid composition, male
Wistar rats (300 g) were prepared for chronic studies in awake animals
by sterile surgical implantation of catheters (Silastic) in the left
femoral artery and vein under short-acting anesthesia (Brevital). The
catheters were exteriorized in the dorsal neck area to prevent
self-induced damage and were flushed regularly with a sterile
hypertonic solution of mannitol and heparin to prevent blockage.
Animals were individually caged and trained to permit sampling and
flushing of the catheters while in a restraining cage for at least 1 wk
before studies. The arterial line was used for blood sampling and blood
pressure monitoring, whereas the venous line was used for the bolus
infusion of LPS (Escherichia coli 0111:B4, 1 mg/kg iv, freshly
diluted in 1 mg/ml 0.9% NaCl; List Biological Laboratories). Some
animals were pretreated with the selective iNOS inhibitor
L-NIL (3 mg/kg ip; BID, Alexis, CA; see Ref. 56) for 3 days
before LPS. This low-dose pretreatment regimen of L-NIL
administration was previously validated by us to avoid change in
systemic blood pressure, renal function, or plasma nitrates.
Furthermore, because L-NIL is an analog of lysine and a
substrate for the Y+ transport system, a high bolus dose
could potentially affect arginine uptake.
Blood samples (80 µl) were collected from the arterial line at timed
intervals in heparinized capillary tubes (Sigma) before and after LPS
infusion. In all experiments, animals were killed by rapid
exsanguination while under anesthesia in accordance with good animal
practice guidelines. Tissue samples were rapidly blotted on absorbent
pads, dissected, and then placed on ice for enzyme studies described
below or snap-frozen in liquid nitrogen and stored at
70°C
to await HPLC analysis as described below. For renal tissue, both
kidneys were decapsulated and further dissected to isolate the cortex.
For liver and cardiac tissue, sections of ~1 g were dissected.
Pulmonary tissue in these studies consisted of one upper and one lower
lobe pooled after removal of large vessels and airways.
Plasma and tissue sampling for HPLC studies.
For each blood sample, the plasma fraction was rapidly separated by
centrifugation, and a 30-µl aliquot was mixed with an equal volume of
10% TCA in 20 mM HCl solution containing 10
4 M
homocysteic acid as an internal standard. Proteins were denatured, and
amino acids were extracted in this mixture for 24 h at 4°C before
high-speed centrifugation to separate the protein pellet from the
supernatant. Samples were stored at
70°C until further processing for HPLC analysis. Processing of snap-frozen tissue for
amino acid extraction involved pulverization of ~100 mg of frozen
tissue using a spring-loaded impact hammer kept frozen with liquid
nitrogen. The frozen pulverized tissue was transferred to Eppendorf
tubes containing 1 ml of 10% TCA in 20 mM HCl to denature the proteins
before lyophilization (thereby eliminating enzyme activity and tissue
water content). Samples were then resuspended in 250 µl double
distilled H2O, and the amino acids were
extracted for 24 h at 4°C. After centrifugation at high speed to
precipitate denatured proteins, the protein pellet was set aside for
quantification, and the extract was stored at
70°C. The
tissue water content of each organ was established by determining a
wet-to-dry ratio and protein content from separate samples of each organ.
Fluorescence detection of amino acids.
For HPLC separation of amino acids, the same basic steps were used to
prepare all samples for elution. The sample supernatants were
transferred to a 10,000 molecular weight spin filter (Millipore) for
further purification and then were extracted three times with hydrated
ethyl ether to remove all traces of TCA and lipids.
Plasma, tissue extracts, and appropriate known standards were then
derivatized for fluorescence detection of primary and secondary amine
groups with N-hydroxysuccinimidyl-6-aminoquinoyl carbamate as
per kit instructions (AccQ tag; Waters). Elution was performed using a Hewlett-Packard 1100 series binary HPLC pump system with a 250-mm 3-µm ODS Hypersil C18 RP column (Hewlett-Packard)
maintained at 45°C. Fluorescence was detected in line using a
Waters 470 detector linked to the data acquisition system. Elution
gradients were loosely based on the AccQ tag kit instructions.
Spectrophotometric detection of nitrites and nitrates.
Systemic NO production was assessed from the plasma concentration of
nitrites and nitrates using the Griess reaction in an automated HPLC
system. Aliquots of known standards and of deproteinated plasma (10 µl) were injected on a column of fine mesh cadmium plated with
magnesium to reduce nitrate to nitrite. Postcolumn mixing with Griess
reagents [1% sulfanilic acid in 5%
H3PO4 and 0.1%
N-(1-naphthyl)ethylenediamide dihydrochloride] in a
heated coil leading to a variable-wave ultraviolet detector enabled
on-line recording of absorbance at 650 nm. The column can be bypassed to determine the ratio of nitrate and nitrite in a sample. Griess products in plasma were virtually all nitrates.
ADC and ODC activity.
Tissue for enzymatic studies was obtained from a separate series of
animals acutely anesthetized to permit intravenous LPS infusion.
Animals were subsequently killed at predetermined time points after LPS
or vehicle bolus infusion (1 mg/kg), as described below. The
preparation of viable renal proximal tubules by rapid mechanical and
enzymatic digestion has been previously reported by us (24), and minor
adaptation was suitable to isolate hepatic cells. ADC and ODC activity
was assessed by the generation of radiolabeled CO2 from
[14C]arginine or ornithine [C-1 labeled,
1.5 × 106
counts · min
1
(cpm) · tube
1, 50-60
mCi/mmol; American Radiolabeled Chemicals] using an aliquot of
hepatic cells or renal proximal tubules [~10 mg in 1 ml DMEM (95% O2, pH 7.4, 100 µM arginine and ornithine, 0.05 mM
pyridoxal phosphate, and 0.5 mM MgSO4)]. Large-bore
test tubes, capped with rubber stoppers and fitted with a metabolic
well (Kontes) containing 300 µl of Solvable (Dupont) as a
14CO2 trapping agent, were used to incubate
cells at 37°C for 60 min. Enzyme activity was terminated by
injecting 100% TCA through the cap after 60 min. Metabolic wells
containing trapped 14CO2 were carefully
transferred to vials containing scintillation fluid for counting. A
standard Lowry test was used to determine the protein content of an
aliquot. ADC and ODC activity is expressed as cpm of CO2
generated in 60 min per milligram protein per cpm added.
iNOS PCR.
In a separate series of rats, two to three rats were killed at 0, 0.5, 1, 2, 4, 6, and 16 h post-LPS to recover the kidneys. RT-PCR analysis
was used to determine iNOS mRNA in total extracts of renal cortex
tissue. Ten micrograms total RNA extract were reverse transcribed with
0.1 µl of 5× buffer, 5 µl dithiothreitol (0.1 M), 10 µl
dNTP (2.5 mM), 0.7 µg oligo(dT), and 1 µg RT in a total volume of
50 µl. The reaction was stopped by heating the sample at 65°C for
10 min. PCR amplification was performed in a reaction volume of 100 µl using a thermal cycler. cDNA was added to the reaction mixture
containing 10 µl of 10× buffer, 2.5 ml dNTP (0.5 mM), 1 µl of
each primer, and 0.5 µl Taq DNA polymerase (1.25 units). The
iNOS primers used are based on the following rat sequence: sense
5'-GCCTCCCTCTGGAAAGA-3' and antisense
5'-TCCATGCAGACAACCTT-3'. Thirty-five cycles were performed
under the following conditions: 95°C for 1 min (denaturation),
54°C for 1 min (annealing), 72°C for 2 min (extension), and
72°C for 7 min (final extension). Aliquots of the PCR products (15 µl) were then separated by gel electrophoresis on a 1% agarose gel
stained with ethidium bromide. Rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to assess total mRNA and normalize iNOS
content of each well. Densitometry evaluation (Scion) of a digitized
image enabled the calculation of iNOS to GAPDH ratios.
Statistics.
ANOVA was used to determine significant differences between paired
values. Most assays were performed in duplicate or were completely
reproduced in the case of HPLC detection, and mean values obtained for
the same test sample were treated as a single value. Unless stated
otherwise, the changes described in RESULTS were
statistically significant (P < 0.05).
 |
RESULTS |
Early and late phase responses.
Systemic blood pressure decreased significantly from 103 ± 4 to 90 ± 6 mmHg within 30 min after intravenous LPS. Pretreatment with
L-NIL completely prevented the effects of LPS on blood
pressure (Fig. 1). A significant increase
in plasma Griess products, an index of NO production, from a baseline
value of 47 ± 12 to 123 ± 32 µM (n = 5) occurred 3 h
post-LPS, whereas no change was observed in either control or
L-NIL-pretreated animals. In three rats, we monitored the
profile of plasma Griess products over 24 h, as represented in Fig.
2. The sublethal dose of LPS used in these experiments caused an increasing accumulation of plasma nitrate for at
least 8 h that returned to normal by 24 h. Accordingly, de novo iNOS
expression as determined by RT-PCR became evident in rat kidney within
60 but not 30 min after LPS, was maximal between 2 and 4 h, and was
normal by 16 h (Fig. 3). Unexpectedly, a
significant amount of iNOS mRNA was observed in control rat kidneys
(i.e., time 0), resulting in a mean GAPDH-to-iNOS ratio of 2.9, a value that subsequently increased 30-fold between 1 and 6 h post-LPS.

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Fig. 1.
Changes in arterial blood pressure after iv lipopolysaccharide (LPS)
with or without
L-N6(1-iminoethyl)lysine
(L-NIL; n = 6 rats; * P < 0.05) vs. control (CTL).
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Fig. 2.
Plasma nitrites increase after iv bolus of LPS, are blocked by
L-NIL, and are normal within 24 h.
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Fig. 3.
Expression of inducible (i) nitric oxide synthase (NOS) mRNA in rat
kidney cortex after LPS. Each lane represents kidney mRNA from an
individual rat killed at the time indicated.
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The range in plasma concentration of arginine and arginine-derived
products spans three orders of magnitude, as indicated on the scale of
each graph in Fig. 4. Arginine in plasma
decreased in both LPS and L-NIL + LPS-treated rats
within 30 min from 134.1 ± 2.9 and 147.5 ± 2.7 µM to 91.1 ± 4.4 and 100.6 ± 3.9 µM, respectively. Plasma arginine levels further
decreased to 60.9 ± 6.5 µM at 180 min and remained low for 6 h in
the LPS group, supporting the concept that arginine uptake is
upregulated by LPS. Plasma ornithine level followed a similar pattern
of change, likely reflecting uptake by the same transporter, but tended
to return to normal after 3 h. Another very rapid response to LPS was
the transient increase in plasma spermidine concentration from 1.74 ± 0.11 to 3.28 ± 0.15 µM in 60 min. Curiously, L-NIL
treatment alone significantly elevated baseline spermidine levels to
3.78 ± 0.13, which subsequently decreased to control values by 120 min after LPS, as if polyamine synthesis and/or transport was not only
initiated but also rapidly switched off. Spermine concentration in
plasma was the lowest of the substances measured (0.25 ± 0.09 µM),
and no significant changes were observed in either experimental
condition.
A number of events appear to occur in a later phase (3-6 h) after
LPS infusion. Plasma citrulline levels increased in the LPS-treated
group from a baseline value of 68.1 ± 2.2 to 86.5 ± 2.8 µM only
after 4 h, an effect completely blocked by L-NIL pretreatment (Fig. 4). This is synchronous with increased nitrate levels in plasma and likely reflects massive NO production in tissue by
iNOS. Plasma putrescine increased significantly in both LPS-treated
groups but only after 3-4 h, suggesting a delayed increase in ODC
activity. Circulating agmatine levels were unchanged in animals treated
with LPS alone but increased significantly after 4 h in the
L-NIL-pretreated group from 2.83 ± 0.35 to a maximum of
4.69 ± 0.21 µM after 6 h. As seen in Fig.
5, a significant increase in ODC and ADC
activity was evident in proximal tubules and liver cells harvested
6 h post-LPS. ODC activity increased 48.79% in the kidney
and 64.48% in the liver, whereas ADC activity increased 27.17 and
31.74%, respectively.

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Fig. 5.
Arginine (ADC) and ornithine (ODC) decarboxylase activity in kidney and
liver cells from rats 6 h post-iv LPS or vehicle; n = 4, * P < 0.05 vs. CTL, y-axis = 103 cpm
14CO2 · mg
protein 1 · h 1
at 37°C from 106 cpm
[14C]arginine or
[14C]ornithine.
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Arginine and related products in tissue.
We determined the ratio of wet to dry tissue weight for each organ
sampled 6 h post-LPS to evaluate potential changes in extracellular volume. No change occurred in either renal, hepatic, or cardiac tissue
(wet-to-dry ratio: 2.45 ± 0.03, 1.79 ± 0.01, and 2.19 ± 0.01, respectively). However, the appearance of edema was evident in
pulmonary tissue, with the wet-to-dry tissue ratio increasing significantly from 2.25 ± 0.02 to 2.42 ± 0.04, an effect that was
blocked by L-NIL pretreatment. Calculations based on tissue water and protein content reveal that amino acid concentration can
exceed plasma values by an order of magnitude and differs markedly
among tissue types. For example, in control animals, we estimate that
intracellular arginine concentration in the kidney is ~5 vs. 0.5 mM
in the liver, whereas values for ornithine are 0.8 and 2.0 mM,
respectively. In the absence of a marker for extracellular volume in
these experiments, calculations of intracellular concentration from
these data underestimate actual values, particularly in tissue where
there is potential for edema. Therefore, to enable comparison between
tissue types, values for tissue content are expressed in relation to
tissue protein (nmol/mg). The results depicted in Fig.
6 demonstrate substantial differences in
amino acid composition of the kidney, heart, liver, and lung. In
untreated animals, it is immediately apparent that the kidney contains
substantially more arginine than the other organs tested. Also, a far
greater proportion of arginine with respect to citrulline and ornithine (17.59 ± 0.50, 4.51 ± 0.21, and 3.00 ± 0.48 nmol/mg,
respectively) is maintained in the kidney, whereas in liver ornithine
predominates (0.79 ± 0.10, 1.54 ± 0.16, and 3.84 ± 0.20 nmol/mg,
for arginine, citrulline, and ornithine, respectively). It is also
evident that the organs tested contain more agmatine and polyamines in
relation to arginine than was seen in plasma. Furthermore, unlike
plasma, tissue putrescine content was found to be consistently lowest of the arginine-related metabolites measured.

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Fig. 6.
Changes in tissue arginine and related metabolites after iv LPS with or
without L-NIL; n = 4, * P < 0.05 vs.
CTL. CIT, citrulline; ARG, arginine; AGM, agmatine; ORN, ornithine;
PUT, putrescine; SPD, spermidine; SPM, spermine; prot, protein.
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No change in tissue arginine content could be demonstrated after LPS
treatment in kidney liver or lung despite the evidence of high iNOS
activity at 6 h, but a significant decrease from 4.18 ± 0.13 to 2.88 ± 0.22 nmol/mg did occur in the heart. Also, only a small but
significant elevation in citrulline content was observed in the lung
(2.12 ± 0.13 to 2.86 ± 0.05 nmol/mg) and liver (1.54 ± 0.16 to
2.11 ± 0.09 nmol/mg) of septic rats. Tissue ornithine content of
LPS-treated rats was significantly increased from control values in
kidney (3.00 ± 0.48 to 3.98 ± 0.30 nmol/mg), liver (3.84 ± 0.20 to 5.76 ± 0.39 nmol/mg), and lung (1.16 ± 0.06 to 2.03 ± 0.12 nmol/mg). Baseline agmatine content was similar in all four
tissues tested and only increased significantly in renal tissue from
1.61 ± 0.13 to 2.48 ± 0.08 nmol/mg. In addition, LPS treatment
caused changes in tissue polyamine content that were tissue specific.
In the kidney, spermidine levels increased significantly from 4.74 ± 0.14 to 5.21 ± 0.18 nmol/mg. In the liver, spermine levels increased
from 3.13 ± 0.13 to 3.59 ± 0.17 nmol/mg. In the lung, both
putrescine and spermidine levels increased from 0.46 ± 0.10 to 0.74 ± 0.15 and 4.45 ± 0.13 to 5.47 ± 0.19 nmol/mg, respectively. As
with arginine in cardiac tissue, spermidine and spermine levels in the
heart tended to be lower after LPS treatment (significant for spermine
only, from 1.97 ± 0.12 to 1.56 ± 0.12 nmol/mg). Clearly, LPS
effected major changes in arginine metabolism beyond simply converting
arginine to NO and citrulline.
Pretreatment with the iNOS inhibitor L-NIL in rats
subjected to LPS had significant and unexpected effects on tissue amino acid composition. A consistent trend in tissue from
L-NIL-treated animals was observed whereby agmatine content
was greater than control or LPS treatment alone, as was observed in
plasma. In the kidney, the levels of arginine, citrulline, ornithine,
and agmatine (27.41 ± 1.21, 5.80 ± 0.20, 5.49 ± 0.26, and 2.88 ± 0.15 nmol/mg, respectively) were all significantly greater than
corresponding values for control or LPS treatment alone. The increase
in renal spermidine resulting from LPS alone was abrogated by
L-NIL, and spermine content decreased significantly (3.71 ± 0.33 nmol/mg). In the liver, more citrulline, agmatine, and
ornithine (2.93 ± 0.18, 1.33 ± 0.08, and 7.23 ± 0.52 nmol/mg,
respectively) were detected than in the other experimental groups, but
arginine content was unaffected and remained the lowest of the tissues
tested. Pulmonary content of all the arginine products measured was
greater in L-NIL-treated rats than control with the
exception of spermine (6.00 ± 0.12, 2.66 ± 0.09, 1.61 ± 0.09, 1.05 ± 0.10, 0.73 ± 0.11, and 5.31 ± 0.14 nmol/mg, for arginine,
citrulline, ornithine, agmatine, putrescine, and spermidine,
respectively). In cardiac tissue, L-NIL prevented the
decrease in arginine, spermidine, and spermine caused by LPS (4.11 ± 0.11, 2.18 ± 0.17, and 2.21 ± 0.10 nmol/mg, respectively). As
predicted, the blockade of iNOS activity caused an increase in the
production of other arginine-derived bioactive products.
 |
DISCUSSION |
Early phase effects of LPS.
In characterizing the LPS-induced events with respect to arginine
metabolism, it became clear that functional and physical changes
occurred before evidence of de novo iNOS activity. Arginine and
ornithine concentrations in plasma decreased rapidly and significantly after LPS, whereas that of other substances remained stable, indicating specific uptake. This finding is in accordance with in vitro studies demonstrating increased affinity and transport rates (Michaelis constant and maximal velocity) of the Y+ system after LPS
(3, 20, 55) and other stimuli (11). The data reported herein raise the
question as to why arginine uptake precedes de novo iNOS synthesis and
activity. At least two obvious explanations come to mind. 1)
Arginine delivery for a quiescent substrate limited fast-response NOS.
2) Rapid arginine influx may be part of the cascade leading to
de novo expression of iNOS and related enzymes. As discussed below,
there is supporting evidence in the literature for both hypotheses.
We and others (2, 5, 8, and unpublished observation) have observed that
arginine infusion elicits a number of responses, including NO
production, and that plasma arginine is rapidly restored to normal
levels, supporting the concept that uptake may be rate limiting in
certain tissues in vivo. Recent studies using LPS-treated rats (13),
pigs (14), dogs (19), and rabbits (41) have also demonstrated an early
phase decrease in systemic arterial pressure. Although it is known that
LPS may elicit the release of vasoactive substances other than NO, we
unexpectedly observed that iNOS blockade with L-NIL
completely abrogated this early phase hemodynamic response. How could
L-NIL prevent the early phase hypotension after LPS?
Possibly, L-NIL effects result from nonselective NOS
blockade, yet we did not observe an increase in systemic blood pressure
typical of constitutive endothelial NOS inhibitors. In fact, the
selectivity of L-NIL and the dosage regimen aimed to avoid
such effects. An alternate explanation derives from evidence of
"constitutive" iNOS expression documented in the human airway
(16) and rat kidney (31). Indeed, there are functional results reported
that document an LPS-induced increase in expired NO before increased
expression of any NOS isoform (13, 14, 19, 41). It is worth noting that
no further decrease in blood pressure occurs after 60 min, indicating
that maximal vasodilation is achieved very rapidly and is maintained,
although admittedly de novo iNOS may play a role in the later phase.
Why plasma citrulline and Griess products are not elevated in the early
phase remains to be explained. The amount of citrulline and Griess
products generated might be relatively small with respect to plasma
content or may be converted efficiently within the cell. With respect
to plasma nitrite and nitrates (Griess products), alternate products of
NO (i.e., peroxynitrite) may possibly play a role in early phase
vasoactivity. Regardless of the NOS isoform blocked by
L-NIL, these data demonstrate how the early phase
hypertension after LPS is dissociated from de novo iNOS activity.
Because polyamines are known mediators of gene transcription, it is
tempting to speculate that iNOS induction might require a signal from
another arginine metabolite. It is interesting to observe that
spermidine concentration in plasma rapidly and transiently doubled
after LPS, suggesting a potential link between de novo iNOS induction
and polyamine metabolism. There is supporting evidence for this concept
from reports of rapid changes in polyamine content in vivo (27) and
the regulation of a protein-synthesis initiation factor
by spermidine (15). Furthermore, a counterregulatory mechanism has been characterized in which polyamine metabolites had an
inhibitory effect on iNOS induction (51).
Late phase effects of LPS.
A time lag of 2-3 h post-LPS occurred before the awake rats
displayed visible signs of septicemia (lethargy, shivering,
piloerection). This corresponds temporally to ~1 h after the
appearance of de novo iNOS mRNA in tissue, most likely due to
translation and processing. Accordingly, substantial increases in
plasma citrulline and Griess products occur 3-4 h post-LPS,
indicating large-scale NO production. It is worth noting that no
further decrease in plasma arginine was detected at this critical time;
in fact there appeared to be a trend toward an increase, suggesting a
highly coordinated adaptation to maintain plasma arginine
concentration. Alternatively, stable plasma arginine levels may
indicate a shift away from dependence on uptake from plasma. Reports in
the literature have addressed this issue. For example, tissue-specific
expression of mRNA for argininosuccinate synthase and lyase was
cosynchronous with respect to iNOS (32), and a decrease in arginase was
demonstrated that coincided with increased iNOS (10). The impact of
L-NIL on the events at this time period is evident from the
absolute abrogation of citrulline and Griess product accumulation in
plasma and a gradual return to normal of plasma arginine and ornithine
within 6 h. Further experiments will be required to determine if the maintenance of low plasma arginine in the later phase results from high
iNOS activity or as a mechanism of regulating uptake-dependent NO production.
Further evidence of a late phase change in arginine metabolism may be
gleaned from plasma measurements of agmatine and putrescine, the
products of arginine and ornithine decarboxylation. Plasma putrescine
increased rapidly between 2 and 3 h after LPS, an effect that was
amplified by iNOS blockade, whereas agmatine increased in the
L-NIL group only. Although the source of these circulating substances cannot be determined from these experiments, we were able to
demonstrate an increase in both ADC and ODC activity in freshly
harvested liver and kidney 6 h after LPS. Together these data suggest
that ADC and ODC activity may depend on substrate availability and/or
regulation by NO itself. With respect to the latter, in vitro studies
have shown that NO inactivates soluble ODC (39) and that membrane-bound
ADC (38) decreases 18-24 h after iNOS induction.
Arginine disposition in tissue.
The differences in the amino acid composition of the kidney, liver,
heart, and lung reflect the specialized functional role of arginine
products in these organs. Accordingly, we observed arginine levels in
the kidney far in excess of other substances and a predominance of
ornithine over citrulline and arginine in the liver. Such differences
should be taken into account when comparing whole cell enzymatic
activity from different tissue types using competitive inhibitors.
Estimations based on tissue amino acid and water content reveal a large
cell to plasma gradient for arginine and virtually all related
products. In light of this, it is particularly difficult to imagine
that arginine availability would be rate limiting for any enzyme in
these organs unless there is intracellular sequestration. After LPS
alone, little or no difference in arginine content of kidney, liver,
and lung was observed, yet, in the presence of L-NIL,
increases in intracellular arginine above control values indicate that
compensatory mechanisms help sustain iNOS. Clearly, a high degree of
tissue-specific adaptation involving varying degrees of transport and
synthesis serves to maintain intracellular and plasma arginine levels.
Depletion of arginine in the heart may have resulted from a
compensatory increase in cardiac output and energy expenditure (link
between the urea cycle and tricarboxylic acid cycle in the
mitochondria) or a greater dependency on arginine uptake. Studies in ex
vivo cardiac tissue responses to LPS have demonstrated some unique
characteristics such as a lack of complete urea cycle (45, 30) and mRNA
translation but no iNOS (25) activity.
In sharp contrast to observations in plasma, citrulline in the kidney,
lung, and liver increased after LPS in L-NIL-treated rats.
This suggests that an increase in urea cycle enzymes, concomitant with
iNOS activation, promotes the generation of citrulline from ornithine. Normally, during iNOS activity the arginase
pathway of ornithine synthesis would be inhibited by NO (7, 44) but not
during iNOS blockade. The substantial tissue citrulline concentration and upregulation of urea cycle enzymes should therefore be taken into
consideration in studies purporting to measure iNOS activity by the
generation of citrulline. The induction and blockade of iNOS resulted
in some significant, tissue-specific effects on polyamine levels that
could potentially exert functional effects. As noted previously, the
exact role of polyamines has not been fully elucidated but includes
mediation of critical cell functions such as replication and
senescence. In light of this, it should be noted that the impact of
sepsis and iNOS blockade could have substantially different effects in
the elderly, since polyamine metabolism is known to change with age
(12). In tissue, putrescine is maintained at substantially lower levels
than ornithine or the polyamines in plasma, indicating that transport
out of the cell may serve to regulate polyamine synthesis. Consistent
with changes in plasma, agmatine levels increased in all four organs tested when iNOS activity was blocked. This again raises the
possibility that L-NIL treatment increased agmatine
production by abrogating inhibitory effects of NO, shunting arginine or
as a feedback response to elevated ODC activity.
We conclude 1) hypotension and uptake of plasma arginine is
evident within 30 min of LPS administration; 2) evidence of de novo iNOS activity first appears 3-4 h after LPS; 3) the
early phase hypotension after LPS is inhibitable by L-NIL;
4) changes in plasma content of ornithine, agmatine, and
polyamines occur before and after de novo iNOS activity; 5) LPS
causes increased ADC and ODC activity, and blockade of iNOS results in
increased agmatine and polyamine levels; 6) tissue arginine
contents and the composition of arginine-derived products differ
markedly among organs; and 7) substantial changes occur in a
tissue-specific fashion to the amino acid composition of the kidney,
lung, liver, and heart as a result of iNOS induction by LPS and
blockade with L-NIL. Further studies will be needed to
elucidate the time course and functional impact of redirected arginine
metabolism with particular attention to specific organs and tissues.
 |
ACKNOWLEDGEMENTS |
We acknowledge support of the following: National Institute of
Diabetes and Digestive and Kidney Diseases Grant DK-28602, NKF of
Southern California for D. Schwartz's fellowship support, and the San
Diego Veterans Medical Research Service.
 |
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: M. J Lortie,
Div. Nephrology/Hypertension, 3350 La Jolla Village Dr., San Diego, CA
92161 (E-mail: mlortie{at}UCSD.edu).
Received 6 August 1999; accepted in final form 22 December 1999.
 |
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