(Received for publication, September 21, 1995; and in revised form, January 2, 1996)
From the
Cytokine-dependent production of nitric oxide (NO) by rat
cardiac myocytes is a consequence of increased expression of the
inducible isoform of nitric oxide synthase (iNOS or NOS2) and, in the
presence of insulin, depresses the contractile function of these cells in vivo and in vitro. Experiments reported here show
that L-lysine, a competitive antagonist of L-arginine
uptake, suppressed NO production (detected as nitrite accumulation) by
interleukin (IL)-1 and interferon (IFN)
-pretreated cardiac
myocytes by 70%, demonstrating that NO production is dependent on L-arginine uptake. Cardiac myocytes constitutively exhibit a
high-affinity L-arginine transport system (K
= 125 µM; V
= 44 pmol/2
10
cells/min). Following a 24-h exposure to IL-1
and IFN
,
arginine uptake increases (V
= 167 pmol/2
10
cells/min) and a second low-affinity L-arginine transporter activity appears (K
= 1.2 mM). To examine the molecular basis
for these cytokine-induced changes in arginine transport, we examined
expression of three related arginine transporters previously identified
in other cell types. mRNA for the high-affinity cationic amino acid
transporter-1 (CAT-1) is expressed in resting myocytes and steady-state
levels increase by 10-fold following exposure to IL-1
and
IFN
. Only cytokine-pretreated myocytes expressed a second
high-affinity L-arginine transporter, CAT-2B, as well as a
low-affinity L-arginine transporter, CAT-2A. In addition,
insulin, which potentiated cytokine-dependent NO production independent
of any change in NOS activity, increased myocyte L-arginine
uptake by 2-fold and steady-state levels of CAT-1, but not CAT-2A or
CAT-2B mRNA. Thus, NO production by cardiac myocytes exposed to
IL-1
plus IFN
appears to be dependent on the co-induction of
CAT-1, CAT-2A, and CAT-2B, while insulin independently augments L-arginine transport through CAT-1.
The inducible isoform of nitric oxide synthase (iNOS or NOS2) ()is expressed in a wide variety of cell types including
neonatal and adult cardiac myocytes after exposure to inflammatory
mediators(1, 2, 3, 4, 5, 6) .
Nitric oxide (NO) production by cardiac myocytes in vitro decreases spontaneous beating rate (a negative chronotropic
effect) (5, 7) and the velocity and extent of
shortening (a negative inotropic
effect)(6, 7, 8, 9, 10, 11, 12) ,
and accelerates the velocity of re-lengthening (a positive lusitropic
effect)(13, 14) . We recently demonstrated that
cytokine-induced NO production by adult rat ventricular myocytes
(ARVM), and its effect to diminish the inotropic responsiveness of
these cells in vitro to the
-adrenergic agonist
isoproterenol, was insulin-dependent (15) . The observation
that this effect of insulin was not a consequence of changes in NOS
activity suggested that insulin was affecting myocyte NO generation by
additional mechanisms that were independent of the extent of increased
expression and activity of NOS2 protein. Potential additional sites for
regulating cellular NO production include the availability of
intracellular arginine and NOS co-factors. Changes in arginine supply
could result from changes in uptake and/or de novo synthesis.
Cellular NO production by NOS2 is dependent on extracellular
arginine both in vitro and in
vivo(16, 17, 18, 19) , and
vascular hyporesponsiveness to vasoconstrictor agonists after in
vivo lipopolysaccharide exposure also appears to be dependent on
extracellular arginine(20) . Arginine is obtained from
exogenous sources via a plasma membrane cationic amino acid transport
system termed ``system
y''(21, 22) . The transport
activity of system y
is characterized by a
high-affinity for cationic amino acids, sodium independence, and
stimulation of transport by substrate on the opposite (trans) side of
the membrane(21, 22) . Two transporters exhibiting
y
system properties were cloned from the mouse and
were termed mouse cationic amino acid transporter-1 and -2B (CAT-1 and
CAT-2B)(23, 24, 25, 26, 27, 28) .
CAT-1 is widely expressed in murine tissues, whereas CAT-2B has been
identified only in activated murine macrophages and
lymphocytes(25, 28) . A third member of the cationic
amino acid transporter family, cloned from murine hepatocytes (CAT-2A),
was distinguished by its lower affinity for cationic amino acids and
insensitivity to trans-stimulation (i.e. absence of the
``y
'' phenotype)(29, 30) .
Consistent with previous reports that failed to detect y
activity in hepatocytes(22) , CAT-2A has been detected
only in the liver of adult rodents, whereas the high-affinity CAT-1 was
not(24, 29) . Changes in the activity of these
transporters could potentially alter NO production.
Arginine can also be generated within cells from intracellular protein degradation and by the endogenous synthesis of arginine(31) . Many NO-producing cell types, including murine macrophages and bovine aortic endothelial cells, are capable of synthesizing arginine from citrulline by the sequential action of argininosuccinate synthetase and argininosuccinate lyase(32, 33) . Expression of argininosuccinate synthetase is increased along with NOS2 in macrophages and vascular smooth muscle cells following exposure to soluble inflammatory mediators (34, 35) and is associated with increased synthesis of arginine from citrulline(32) .
Another potential site for regulation of
cellular NO production by cytokines and insulin is the availability of
tetrahydrobiopterin (BH), an essential NOS
co-factor(3) . GTP cyclohydrolase I, the rate-limiting enzyme
for the de novo synthesis of BH
, is co-induced
with NOS2 by cytokines in a variety of cell types, including cardiac
myocytes(6, 36, 37, 38) . Inhibitors
of GTP cyclohydrolase I limited the production of NO by rat aortic
smooth muscle cells in vitro following NOS2 induction,
indicating that BH
availability also may limit NO
production in cytokine-pretreated cells(39) .
In the
experiments described here, we addressed the mechanisms by which
IL-1 and IFN
, and insulin regulate L-arginine
availability. To this end, we measured the dependence of
cytokine-induced NO production on L-arginine transport in rat
cardiac myocytes and correlated this with mRNA levels for the three
cloned cationic amino acid transporters. We also determined whether
cytokine- and insulin-mediated augmentation of NO production in these
cells is associated with changes in levels of mRNA coding for
argininosuccinate synthetase and argininosuccinate lyase, enzymes for
the de novo synthesis of arginine, or for GTP cyclohydrolase
I, the rate-limiting enzyme for BH
synthesis. These data
support the conclusion that L-arginine transport into cardiac
myocytes is rate-limiting for NO production by cardiac myocytes in the
presence of insulin and these cytokines.
Neonatal rat ventricular myocytes (NRVM) were
isolated from 1-day-old Sprague-Dawley pups as described
previously(42, 43) . Briefly, neonatal cardiac
ventricular tissue was excised aseptically and then digested with 0.1%
trypsin in Hanks' balanced salt solution (Life Technologies,
Inc.) overnight at 4 °C. Ventricular cells were then recovered by
repeated digestions of the tissue in 10 ml of 0.1% collagenase in
Hanks' solution at 37 °C in a shaking water bath. The
supernatants collected from each digestion were centrifuged at 1000 rpm
for 4 min (4 °C). The pellets were then resuspended in ice-cold
Hanks' solution, pooled, and centrifuged at 1000 rpm for 4 min (4
°C). Cells were resuspended in DMEM supplemented with 7% fetal calf
serum (Life Technologies, Inc.) and subsequently underwent two
preplatings of 75 min each to minimize nonmyocyte contamination to less
than 5% of the enriched myocyte population(43) . Nonadherent
cells were counted with a hemacytometer and plated at a density of 1000
cells/mm. After 24 h, the culture medium was changed to
serum-free DMEM supplemented where indicated with 100 nM insulin. All experiments were performed 24 h later at which time
spontaneous contractile activity was evident.
Total RNA was isolated from adult and
neonatal ventricular myocytes using the method of Chomczynski and
Sacchi(45) . RNase protection analysis was performed as
described(46) . Following gel purification of the probes,
hybridization reactions were performed with 20 µg of total RNA in
50% formamide for 12 h at 50 °C using 2 10
cpm
per reaction of the radiolabeled antisense RNA transcripts, except for
the 18 S riboprobe where 10
cpm per reaction was
co-hybridized with all samples. Samples were then digested with
ribonuclease A and T1 (Boehringer Mannheim) and analyzed on 8%
denaturing polyacrylamide gels, with adjacent RNA size markers
(Ambion), followed by autoradiography. Total RNA from murine macrophage
and liver was also analyzed by RNase protection on initial gels that
verified the presence of protected fragments of the predicted size for
the rat.
Figure 1:
Dependence of cytokine-induced nitrite
production and its regulation by insulin on L-arginine uptake
by cardiac myocytes. 24-h nitrite accumulation in medium (upper
panel) conditioned by neonatal rat ventricular myocytes treated
with IL-1 and IFN
(open bars) or the combination of
IL-1
, IFN
, and insulin (black bars), in the absence
and presence of 10 mML-lysine. Nitrite accumulation
was undetectable in medium conditioned by control myocytes or myocytes
exposed to insulin alone. Following 24-h incubation with the indicated
treatments, uptake of L-[
H]arginine (100
µM) was measured for 5 min in the absence and presence of
10 mML-lysine (lower panel). Each point is
the mean ± S.E. from six repetitions and is from a single
experiment that was representative of four separate experiments (*, p < 0.001 versus other groups;**, p <
0.001 versus control; #, p < 0.001 versus absence of L-lysine).
The dependence of NO production in intact
myocytes on L-arginine transport was studied by incubating
neonatal cardiac myocytes for 24 h with IL-1 and IFN
in the
presence and absence of L-lysine, a cationic amino acid that
competes with L-arginine for transmembrane
transport(21) . Insulin and the combination of IL-1
and
IFN
were both observed to increase L-[
H]arginine (100 µM)
uptake 2-fold compared to control (p < 0.001), and the
effects on L-arginine uptake of insulin and the cytokines were
additive (Fig. 1, lower panel). The 100 µM concentration of L-arginine was chosen as it is the
approximate mammalian plasma concentration for this amino
acid(53) . A concentration of L-lysine (10
mM) was selected that was determined to inhibit myocyte L-arginine (100 µM) uptake by >95%. The
addition of L-lysine to the medium reduced cytokine-induced
nitrite production by two-thirds (Fig. 1, upper panel),
whereas D-arginine (10 mM) had no effect on nitrite
production (data not shown). Addition of L-lysine did not
completely abolish the effect of insulin to augment cytokine-induced
nitrite production, but it did lead to a 15% greater reduction in
lysine-inhibitable (uptake-dependent) nitrite in the cytokine plus
insulin group when compared to cytokines alone.
Consistent with
previous observations, as shown in Fig. 2, the combination of
IL-1 and IFN
(but not insulin) produced comparable increases
in maximal NOS activity at 24 h in cellular homogenates of neonatal and
adult cardiac myocytes above low levels of baseline activity, as
measured by the rate of conversion of L-[
H]arginine to L-[
H]citrulline in the presence of
excess substrate and co-factors. In contrast to its effects on nitrite
production by intact cardiac myocytes (Fig. 2), the 24-h
incubation of cells with L-lysine (10 mM) added to
cytokines did not diminish maximal NOS activity in myocyte homogenates.
The addition of the NOS inhibitor L-N-monomethylarginine (1 mM) to the enzyme
assay buffer decreased maximal NOS activity close to background levels.
Figure 2:
NOS2 activity in cytokine-treated cardiac
myocytes. Whole-cell extracts from neonatal (A) and adult (B) rat ventricular myocytes were assayed for the enzymatic
conversion of L-[H]arginine to L-[
H]citrulline as described under
``Experimental Procedures.'' Cells were incubated for 24 h in
control medium or in medium containing IL-1
and IFN
alone, or
in combination with insulin (100 nM) in the absence or
presence of L-lysine (10 mM). Extracts of the cells
treated with the combination of cytokines and insulin were also
incubated in either a reaction buffer alone or in a reaction buffer
supplemented with 1 mML-N-monomethylarginine (L-NMMA). Data are means ± S.E. from two
experiments (each in triplicate). *, p < 0.01 versus control or L-N-monomethylarginine.
Figure 3:
Time course of L-arginine uptake
by cytokine- and insulin-treated cardiac myocytes. Neonatal rat
ventricular myocytes were exposed to either control medium alone for 24
h (), to control medium containing 4 ng/ml rhIL-1
and 500
units/ml rmIFN
(
), to medium supplemented with 100 nM insulin (
), or the combination of cytokines and insulin
(
). The uptake of 100 µML-[
H]arginine was then initiated as
described under ``Experimental Procedures.'' The data shown
are from a representative experiment (repeated twice with similar
results). Each point represents mean ± S.E. from six replicates.
The S.E. is less than 8.0 pmol/2
10
cells on those
data points where the S.E. bars are not
visible.
To characterize the kinetics of L-arginine transport in ventricular myocytes, saturable uptake
of L-arginine (0-5 mM) was measured. The plot
of uptake of L-arginine as a function of the extracellular L-arginine concentration is shown in Fig. 4. A
high-affinity transporter having a K of 125
± 19 µM and a maximum transport velocity (V
) of 44 ± 2.4 pmol/2
10
cells/min was present in control cells (Fig. 4, A and B). Following a 24-h exposure to IL-1
and
IFN
, arginine uptake increased (V
, 167
± 22 pmol/2
10
cells/min; Fig. 4C) and a second low-affinity transporter activity
appeared (K
= 1.2 mM), in addition
to the high-affinity transport activity (K
= 54 µM), as is evident from the biphasic
Eadie and Hofstee plot in Fig. 4D. This suggested that
there was cytokine regulation of the cationic amino acid transporters,
given the previous report of a low-affinity (CAT-2A) member of this
family(29, 30) .
Figure 4:
Concentration dependence of L-arginine uptake by cardiac myocytes. A, uptake of L-[H]arginine by control neonatal rat
ventricular myocytes was measured for 5 min over a range of
concentrations (0.03-750 µM). The data shown are
from a single experiment that is representative of three separate
experiments. Each point is the mean ± S.E. from 12 replicates. B, Eadie-Hofstee transformation of the data from A gave a K
of 125 µM and
a V
of 44 pmol/2
10
cells/min. C, the effect of 24-h pretreatment with
IL-1
and IFN
on the uptake of L-[
H]-arginine by neonatal rat
ventricular myocytes. Uptake of L-arginine was measured for 5
min over a range of L-arginine concentrations (30 nM to 5 mM). A 24-h exposure to IL-1
and IFN
increased both the K
to 655 ± 233
µM and V
to 167 ± 22 pmol/2
10
cells/min. Each point shown is the mean ±
S.E. from 12 replicates from a single experiment that was
representative of three separate experiments. D, Eadie-Hofstee
transformation of data from C demonstrated both a high- and a
low-affinity transport component for L-arginine uptake; the
high-affinity K
is 54 µM and V
of 56 pmol/2
10
cells/min,
and the low-affinity K
is 1.2 mM with a V
of 213 pmol/2
10
cells/min.
The mRNA for the high-affinity arginine transporter,
CAT-1, was expressed in control cells as shown in Fig. 5.
Treatment of neonatal cardiac myocytes with IL-1 and IFN
for
24 h increased steady-state CAT-1 mRNA levels, normalized to 18 S mRNA,
by 10-fold as determined by densitometry. Insulin independently
increased the expression of CAT-1 transcript and amplified the
induction of CAT-1 by IL-1
and IFN
by 5-fold in both neonatal
and adult cardiac myocyte phenotype. An independent effect of IL-1
and IFN
to induce CAT-1 by at least 2-fold was also observed in
separate experiments in adult cardiac myocytes (data not shown).
Figure 5:
Regulation of CAT-1 mRNA by cytokines and
insulin in cardiac myocytes. Total RNA from primary cultures of
neonatal and adult rat ventricular myocytes was isolated following a
24-h incubation in control medium alone, or following treatment with
IL-1 and IFN
, insulin, or the combination of IL-1
,
IFN
, and insulin. CAT-1 mRNA was detected by RNase protection
analysis. DNA sequencing reactions or a 100-bp RNA ladder (not shown)
were used to determine fragment sizes. Correction for differences in
loading of samples was performed by co-hybridizing with a riboprobe for
18 S ribosomal RNA. This experiment was repeated five times with
similar results. Autoradiograph exposure time was 24 h for CAT-1 and 3
h for 18 S.
The
mRNA coding for CAT-2B, a high-affinity cationic amino acid transporter
originally described in activated macrophages and
lymphocytes(25, 28) , was only consistently detected
in myocytes after a 24-h exposure to IL-1 and IFN
(Fig. 6). Note that in contrast to CAT-1, insulin did not alter
the levels of mRNA for CAT-2B following exposure to the cytokines.
Figure 6:
Regulation of CAT-2B and CAT-2A mRNA by
cytokines in cardiac myocytes. Primary cultures of neonatal rat
ventricular myocytes were incubated for 24 h in control medium, or
treated with IL-1 and IFN
, insulin, or the combination of
IL-1
, IFN
, and insulin. Total RNA was analyzed by RNase
protection with DNA sequencing reactions or a 100-bp RNA ladder (not
shown) used to determine fragment sizes. Correction for differences in
loading of samples was performed by co-hybridizing with a riboprobe for
18 S ribosomal RNA. This experiment was repeated four times with
similar results. Autoradiograph exposure time was 3 weeks for CAT-2B
and CAT-2A and 6 h for 18 S.
The expression of the low-affinity transporter CAT-2A had previously
been identified only in hepatocytes(29) . However, cardiac
myocytes were also observed to express CAT-2A mRNA (Fig. 6), but
only consistently following a 24-h treatment with IL-1 and
IFN
. This paralleled the induction by cytokines of a low-affinity L-arginine transport system as assessed by kinetic studies (Fig. 4, C and D). As was noted for CAT-2B,
insulin alone or in combination with cytokines did not appear to have
an independent effect on the levels of mRNA for CAT-2A.
Figure 7:
Effect of cytokines and insulin on mRNA
levels for NOS2, argininosuccinate synthetase, argininosuccinate lyase,
and GTP cyclohydrolase I. Northern blots of total RNA from neonatal rat
ventricular myocytes were analyzed as described under
``Experimental Procedures.'' Myocytes were treated for 24 h
with control medium alone or medium supplemented with insulin (100
nM) in the presence and absence of IL-1 and IFN
.
Equal loading of samples was confirmed by hybridizing the same membrane
to a labeled oligonucleotide probe for 18 S ribosomal RNA. These blots
are representative of three independent experiments. Autoradiograph
exposure time was 24 h for all samples, except 18 S, which was exposed
for 5 min.
These results indicate that the increase in sarcolemmal L-arginine transport that accompanies the induction of NOS2 by
the cytokines IL-1 and IFN
in rat cardiac myocytes is
associated with increased expression of the high-affinity cationic
amino acid transporter CAT-1, and co-induction of at least two
additional transporters, CAT-2A and CAT-2B. Approximately two-thirds of
cytokine-induced NO production (measured as nitrite released) was
dependent on the transport of extracellular L-arginine, as
determined by the ability of L-lysine to competitively inhibit L-arginine uptake and nitrite production by cardiac myocytes.
Insulin, which independently increased CAT-1 mRNA levels and L-arginine transport in control myocytes, also enhanced L-arginine transport and nitrite production in
cytokine-pretreated cells. Whenever possible technically, experimental
protocols were performed in both neonatal and adult ventricular myocyte
primary cultures. No qualitative differences were observed between
these two myocyte phenotypes with regard to the cytokine- and/or
insulin-induced effects described here. Specifically, there were no
qualitative differences in NOS activity, nitrite accumulation, arginine
uptake, and CAT-1 mRNA expression.
The enzymatic activity of NOS2 in
homogenates of most cell types in which it has been examined appears to
be regulated mainly at the transcriptional level, although
post-transcriptional and post-translational regulatory mechanisms have
been described(54) . However, the observation that L-lysine diminishes cytokine-induced NO production by intact
cells indicates that L-arginine transport is limiting in NO
synthesis in cardiac myocytes. This dependence of myocyte NO generation
on extracellular L-arginine has also been observed in
activated murine macrophages (16, 17, 55) and
in cytokine-pretreated rat vascular smooth muscle
cells(18, 56) . However, the specific cellular
mechanisms responsible for increased arginine transport were unknown.
In studies of the kinetics of nitrite production by NOS2 in activated
macrophages over a range of extracellular L-arginine
concentrations, the K for L-arginine in
intact cells was 73 to 150 µM(16, 17) .
This value in intact cells is significantly greater than the K
for L-arginine that has been reported
for the isolated enzyme (57) and is close in magnitude to both
the plasma arginine concentration (100 µM) (53) and the K
reported for the
high-affinity L-arginine transporters CAT-1 and
CAT-2B(24, 25, 26, 30) . These
observations, combined with the data in this report, indicate that L-arginine transport may be an important regulatory mechanism
for determining the rate of NO production by NOS2 in cardiac myocytes
and other cell types as well.
The transport properties of CAT-1,
which is constitutively expressed in neonatal and adult cardiac
myocytes, and the cytokine-inducible CAT-2B transporter, have
previously been identified to be characteristic of the y amino acid transporter
phenotype(24, 25, 26, 27, 30) .
CAT-1 mRNA is constitutively expressed in a variety of tissues, with
the exception of the liver(24) , but it has not previously been
shown to be regulated by inflammatory cytokines. This enhanced
expression of CAT-1 and NOS2 by cytokines in cardiac myocytes may have
added significance due to the widespread tissue distribution of CAT-1,
and the growing number of cell types in which NOS2 induction has been
observed. Expression of CAT-2B was initially detected in activated
murine macrophages and lymphocytes(25, 28) , and the
demonstration of its expression in cytokine-pretreated cardiac myocytes
indicates that it also has a wider tissue distribution than original
reports had indicated.
In addition to these high-affinity cationic
amino acid transporters, the kinetic studies reported here show that
cytokine-pretreated cardiac myocytes also exhibit induction of a
low-affinity uptake system for L-arginine that coincided with
the expression of CAT-2A mRNA. CAT-2A has previously been shown to have
the same specificity for cationic amino acids as CAT-1, but with at
least a 10-fold greater K and less sensitivity to
trans-stimulation(29, 30) . CAT-2A had been thought to
be constitutively expressed only in hepatocytes, where it was presumed
to mediate the uptake of cationic amino acids from the portal
circulation after a protein meal(29) . The regulation of CAT-2A
by cytokines has also been observed in murine macrophages. (
)However, the expression of the low-affinity cationic amino
acid transporter in response to cytokines in neonatal cardiac myocytes
(or any cell type other than hepatocytes) may be of little physiologic
significance given that the high-affinity CAT-1 and CAT-2B transporters
would mediate most of the uptake of L-arginine at usual
mammalian plasma concentrations of this amino acid.
In cardiac
muscle, as in many other cell types, insulin regulates a variety of key
metabolic functions including transmembrane amino acid transport and
protein turnover(58, 59) . While the transport of
small aliphatic amino acids into cells by the sodium-dependent amino
acid transporter System A has been shown to be up-regulated by insulin
in a wide range of cell types(60) , the stimulation of cationic
amino acid transport by insulin has not previously been documented.
Interestingly, L-arginine has long been known to regulate
insulin secretion by pancreatic islet cells, an effect that may
be mediated by NO(61, 62) . In cardiac myocytes, we
observed that insulin-dependent L-arginine uptake was
associated with enhanced expression of CAT-1 mRNA. Wu et al.(63) also reported that insulin increases the expression
of the ecotropic retrovirus receptor, now known to be identical to
CAT-1 (24, 26) , in FTO2B rat hepatoma cells although
these authors did not examine the transport of cationic amino acids.
In cardiac myocytes, the effect of insulin on L-arginine
uptake and CAT-1 mRNA levels was additive to that of IL-1 and
IFN
. This effect of insulin to increase arginine transport
contributed, in part, to increased cytokine-induced nitrite production
in cardiac myocytes, although it appears likely that insulin may
potentiate cellular NO production by additional mechanisms that are
independent of NOS activity. This provides one possible mechanism for
our previous observation that insulin is required for the impaired
inotropic response of ARVM to the
-adrenergic agonist
isoproterenol following the induction of NOS2 in
vitro(15) , and for the previously reported negative
inotropic effect of insulin in an in vivo canine model of
sepsis(64) . Furthermore, enhanced L-arginine uptake
into vascular smooth muscle cells, which are known to express NOS2, may
contribute to the insulin-induced hypotension in vivo in the
canine sepsis model(65) .
The additive effects of insulin
and IL-1 plus IFN
to increase CAT-1 expression distinguish
this transporter from the glucose transport system, in which cytokines
such as IL-1 and tumor necrosis factor
, as well as
lipopolysaccharide, impair insulin-mediated glucose
uptake(66, 67) . In vitro exposure to tumor
necrosis factor
or IFN
have been shown to suppress
insulin-induced tyrosine phosphorylation of the insulin receptor and
insulin receptor substrate-1(68, 69) , glucose
uptake(64) , and expression of the glucose transporters GLUT4
and GLUT1(70, 71) . This suggests post-receptor
divergence of insulin signaling for the CAT-1 and glucose transport
systems. Downstream divergence may occur at the ras-raf
mitogen-activated protein kinase and phosphoinositide 3-kinase
pathways, since both pathways are activated by insulin, but the latter
has been shown to be required for glucose uptake via the
insulin-sensitive GLUT4 glucose transporter and is independent of
activation of mitogen-activated protein
kinase(72, 73) . The additive effects of insulin and
IL-1
plus IFN
on CAT-1 expression is consistent with this
explanation since the IL-1
plus IFN
also activates p44/p42
mitogen-activated protein kinase (ERK1/ERK2) in cardiac
myocytes(74) .
The regulation of CAT-1-mediated L-arginine transport by insulin may have broader implications
for the cardiovascular system as this transporter is constitutively
expressed in a wide variety of tissues(24, 75) , and
y transport activity has been demonstrated both in
endothelial cells (76, 77) and vascular smooth muscle
cells(55, 78) . Insulin has been demonstrated to cause
vasodilation that is NO-dependent(79, 80) , although
the mechanism(s) by which it stimulates NO release have been unclear.
It is possible that insulin-mediated L-arginine transport also
could potentially contribute to the regulation of the generation of NO
by the endothelial constitutive NO synthase (ecNOS, NOS3) and that
abnormalities in insulin-induced L-arginine transport could
lead to altered vascular function and hypertension as in
non-insulin-dependent diabetes mellitus. A limitation of NO release
caused by decreased L-arginine transport provides one possible
explanation for the improvement in endothelium-dependent vasodilation
with the systemic administration of L-arginine producing
plasma concentrations of L-arginine far in excess of the K
for the purified NO
synthase(81, 82) .
In addition to enhancing L-arginine transport, inflammatory cytokines and/or insulin
could also affect the de novo synthesis of L-arginine
within cardiac myocytes. The expression of mRNA for argininosuccinate
synthetase, the rate-limiting enzyme in arginine synthesis, was induced
in cardiac myocytes by IL-1 and IFN
, as has been previously
reported in other cell types(34, 35) . In contrast,
argininosuccinate lyase mRNA was constitutively expressed in these
cells and was unaffected by these cytokines. Insulin, either alone or
in combination with cytokines, had no effect on mRNA levels for these
two enzymes that together convert citrulline to arginine. Previous
studies of cultured hepatocytes also have shown that insulin alone has
no effect on the mRNA levels or on the activities of either
argininosuccinate synthetase or argininosuccinate lyase(83) ,
and the activities of these enzymes are not known to be modulated by
post-translational modifications(31) . Arginase mRNA was
undetectable under any conditions in cardiac myocytes, consistent with
the absence of a complete urea cycle in this cell type(31) .
Therefore, recycling of citrulline to arginine may be one source of
arginine for the approximately one-third of cytokine-induced NO
production that is transport-independent, although it is unlikely that
insulin is mediating its effects through this mechanism.
The
regulation of GTP cyclohydrolase I, the rate-limiting enzyme for
BH synthesis, a necessary co-factor for NOS2 activity, by
cytokines and by insulin were also assessed in cardiac myocytes.
Insulin-induced hypoglycemia in rats had previously been reported to
increase the activity of GTP cyclohydrolase I, the rate-limiting enzyme
in the de novo BH
synthesis pathway, as well as
BH
levels in adrenal cells(84) . However, a direct
effect of insulin on BH
or GTP cyclohydrolase I mRNA levels
has not been addressed. Following exposure to IL-1
and IFN
,
GTP cyclohydrolase I mRNA was co-induced with NOS2 in rat ventricular
myocytes, as we have previously reported(6) , but insulin had
no effect on mRNA levels of either enzyme, with or without IL-1
and IFN
. Thus, increased expression of GTP cyclohydrolase I mRNA
does not appear to be contributing to insulin's action of
augmenting cytokine-induced NO production.
A limitation of the
current study is that arginine uptake rates and arginine
uptake-dependent NO production are only correlated with the expression
of mRNA for CAT-1, CAT-2B, and CAT-2A. Support for this correlation
representing a causal relationship between CAT-mediated L-arginine uptake and NO production in these cells includes
the following. 1) The detection of CAT-2A transcripts, the only low
affinity cationic amino acid transporter identified to date, in
cytokine-pretreated myocytes coincided with the detection of a low
affinity transporter based on L-arginine kinetic studies. 2)
The sodium independence of arginine uptake is consistent with the CAT
family of transporters. 3) Reconstitution experiments in Xenopus oocytes have demonstrated that the dependence of nitrite
production by transfected NOS2 is dependent upon co-transfection and
expression of the CAT transporters. ()4) While other
transport systems have been defined in physiologic
terms(60, 85) , the only other cationic amino acid
transporters cloned and sequenced to date, D2 (86) and
4F2(87) , are unlikely to have contributed to L-arginine uptake in our cells. D2 expression is limited to
the kidney and intestine(86) , and the 4F2 transporter has
one-tenth the activity of the CAT family of transporters and could not
account for the kinetic data in this report(87) .
In summary, cellular NO production by cytokine-pretreated cardiac myocytes is determined not only by the extent of transcriptional induction of NOS2, but also by intracellular arginine availability and is dependent on transmembrane arginine transport. Thus, there are multiple potential sites whereby NO production may be regulated in intact cells, and the actual site of regulation may vary for different cell types. This study indicates that increased L-arginine transport following cytokine exposure correlates with the coordinate induction of CAT-1, CAT-2B, and CAT-2A with NOS2 and that arginine transport is necessary for as much as two-thirds of cellular NO production. Furthermore, the fact that changes in NO production do not always reflect changes in NOS enzyme levels is exemplified by the effect of insulin to induce CAT-1 and stimulate L-arginine transport, which appears to contribute to increased cytokine-induced NO production in cardiac myocytes.