(Received for publication, January 19, 1996; and in revised form, March 12, 1996)
From the
Since recent studies demonstrated that platelet-derived growth
factor (PDGF) induces vascular smooth muscle cell (SMC) proliferation
by stimulating polyamine synthesis, we examined whether the
transcellular transport of L-ornithine, the cationic amino
acid precursor of polyamines, could regulate the mitogenic response of
PDGF. Treatment of SMC with PDGF stimulated DNA and putrescine
synthesis, and this was enhanced further by increasing the
extracellular concentration of L-ornithine. The potentiating
effect of L-ornithine was reversed by the competitive
inhibitor of cationic amino acid transport, methyl-L-arginine,
or by preventing putrescine formation with
-difluoromethylornithine. Cationic amino acid uptake by SMC was
Na
-independent and was mediated by both a high and low
affinity carrier system. Treatment of SMC with PDGF initially
(0-2 h) decreased basic amino acid transport, while longer
exposures (6-24 h) progressively increased uptake. Kinetic
studies indicated that PDGF-induced inhibition was associated with a
decrease in affinity for cationic amino acids, while the stimulation
was mediated by an increase in transport capacity. Endogenous PDGF
released by collagen-activated platelets likewise up-regulated cationic
amino acid transport in SMC. Reverse transcriptase-polymerase chain
reaction detected the presence of mRNA encoding two distinct cationic
amino acid transporter (CAT) proteins, CAT-1 and CAT-2B. Treatment of
SMC with PDGF strongly induced the expression CAT-2B mRNA and modestly
elevated the level of CAT-1 mRNA. These results demonstrate that
PDGF-induced polyamine synthesis and SMC mitogenesis are dependent on
the transcellular transport of L-ornithine. The capacity of
PDGF to up-regulate the transport of L-ornithine by inducing
the expression of the genes for CAT-1 and CAT-2B may modulate its
mitogenic effect by providing SMC with the necessary intracellular
precursor for polyamine biosynthesis.
Excessive proliferation of vascular SMC ()is an
important contributing factor to a number of vascular disease states,
including atherosclerosis, hypertension, and restenosis following
angioplasty(1, 2, 3) . One potent modulator
of SMC replication is PDGF(1, 4) . This cationic
peptide is secreted by activated platelets and macrophages, and by
vascular cells at sites of inflammation and vascular
damage(4) . PDGF has been implicated in the regulation of SMC
proliferation both in vivo and in vitro, but the
mechanism(s) by which PDGF induces SMC growth remains
unclear(4, 5) . Recent studies indicate that the
synthesis of polyamines plays an integral role in the mitogenic
response of arterial SMC to PDGF(6) . PDGF-stimulated SMC
proliferation is preceded by increases in cellular polyamine content,
and inhibition of polyamine formation prevents cell growth.
Furthermore, the exogenous addition of polyamines to SMC mimics PDGF in
stimulating DNA synthesis.
The polyamines putrescine, spermidine, and spermine are naturally occurring aliphatic amines, which are present in all mammalian cells. Putrescine is synthesized from the cationic amino acid L-ornithine via a decarboxylation reaction catalyzed by the enzyme ornithine decarboxylase(7) . Spermidine and spermine are synthesized from putrescine through the sequential addition of a propylamine moiety from S-adenosylmethionine(7) . Early studies indicated that induction of ornithine decarboxylase and increases in polyamine biosynthesis are required for cell growth in response to hormones and growth factors(7, 8, 9) . Although this highly regulated inducible enzyme is often believed to be rate-limiting in the polyamine biosynthetic pathway, the availability of L-ornithine also plays a crucial role in regulating polyamine biosynthesis. Previous studies demonstrate that the steady state level of L-ornithine markedly influences polyamine formation in neoplastic cells(10, 11) . Moreover, exogenous L-ornithine administration further stimulates DNA synthesis in these cells, suggesting that the increase in DNA synthesis results from increased polyamine synthesis due to enhanced L-ornithine substrate availability(12) .
Our laboratory and others have
previously demonstrated that the transport of cationic amino acids,
such as L-ornithine, by vascular SMC is mediated by the system
y carrier(13, 14) . This particular
transport system is characterized by its recognition of cationic amino
acids with high affinity, its Na
independence, and the
ability of substrate on the opposite (trans) side of the
membrane to increase transport activity (15) . Recently, the
genes encoding the proteins responsible for the activity of the system
y
carrier have been cloned and designated as CAT-1,
CAT-2A, and CAT-2B(16, 17, 18, 19) .
CAT-1 was initially identified as an ecotropic retrovirus receptor in
murine fibroblasts (16) and was subsequently shown to be a
basic amino acid transporter in Xenopus oocytes(20, 21) . CAT-2B was first detected in
activated thymocytes and has recently been cloned from
lipopolysaccharide-treated macrophages(17, 18) . Both
CAT-1 and CAT-2B are low capacity transporters which have a high
affinity (K
100 µM) for
cationic amino acids. In contrast, CAT-2A is an alternate splice
variant of CAT-2B that was cloned from murine liver that possesses low
affinity (K
= 2-5
mM) but high transport capacity(19) .
Since polyamine generation is dependent on L-ornithine availability, the present study was designed to determine whether PDGF-induced polyamine synthesis and SMC mitogenesis are dependent on the transcellular transport of L-ornithine. We now report that PDGF up-regulates SMC cationic amino acid transport and induces the gene expression of specific CAT proteins. Furthermore, endogenous PDGF released by activated platelets likewise up-regulates cationic amino acid transport in vascular SMC.
The cDNA (10 µl) was amplified in a 50-µl reaction
volume containing MgCl (2.5 mM), a mixture of
dATP, dTTP, dGTP, and dCTP (each at 0.2 mM), CAT primers (50
pmol each), and Taq DNA polymerase (2.5 units/ml) in standard
reaction buffer. Amplification consisted of 30 cycles of PCR (1 min at
95 °C for denaturing, 1 min at 58 °C for annealing, and 2 min
and 5 min (final cycle) at 72 °C for elongation). Products of PCR
amplification were resolved by agarose gel electrophoresis, stained
with ethidium bromide, visualized on a UV transilluminator, and
photographed. When products of expected size were obtained, they were
subcloned into PCRII plasmids (Invitrogen, San Diego, CA) and sequenced
by the dideoxy chain termination method to confirm their identity (26) .
For use in ribonuclease protection assays, small cDNA
fragments of CAT-1 and CAT-2B were amplified from SMC RNA by RT-PCR, as
outlined above. The forward 5`-TCATCGGTACTTCAAGCGTGG-3` (corresponding
to sense bp 563-583 of CAT-1) and reverse
5`-CTGACTCCTTCACGCCAAGAG-3` (corresponding to antisense bp
737-757 of CAT-1) primers for CAT-1 were used to amplify a 195-bp
fragment and the forward 5`-GGGTGTCTTTCCTCATCGCTG-3` (corresponding to
sense bp 1-21 of CAT-2B) and reverse 5`-CAAAGGTGCCACTCCATGCTC-3`
(corresponding to antisense bp 190-210 of CAT-2B) primers for
CAT-2B were used to amplify a 210-bp fragment. The PCR fragments were
then subcloned into PCRII plasmids and sequenced to confirm their
identity and orientation. Antisense RNA probes for CAT-1 and CAT-2B
were generated in the presence of [P]UTP by in vitro transcription using SP6 RNA polymerase.
Treatment of quiescent cultures of rat aortic SMC with PDGF
(30 ng/ml) for 24 h resulted in an approximate 50% increase in the
incorporation of radioactive thymidine into DNA (Fig. 1A). The simultaneous addition of L-ornithine (0.1-1.0 mM) to the culture media
further increased PDGF-induced [H]thymidine
incorporation in a concentration-dependent manner (Fig. 1A). In the absence of PDGF, however, L-ornithine failed to stimulate thymidine incorporation (data
not shown). The ability of L-ornithine to increase
[
H]thymidine incorporation in PDGF-treated cells
was inhibited by L-NMA (10 mM) and by DFMO (2
mM) (Fig. 1B). The PDGF-induced increase in
DNA synthesis was associated with an increase in the capacity of SMC to
generate the polyamine putrescine from extracellular L-ornithine (Fig. 2). Increasing the extracellular L-ornithine concentration from 50 µM to 1 mM further augmented the capacity of PDGF to stimulate putrescine
formation (Fig. 2). Both L-NMA (10 mM) and
DFMO (2 mM) inhibited PDGF-induced putrescine formation (Fig. 2).
Figure 1: Effect of extracellular L-ornithine on PDGF-induced thymidine incorporation in vascular SMC. A, SMC were treated with PDGF (30 ng/ml) in the presence of various concentrations of L-ornithine (0-1.0 mM) for 24 h. B, SMC were treated with PDGF (30 ng/ml) in media containing L-ornithine (1 mM) for 24 h in the presence of L-NMA (10 mM) or DFMO (2 mM). Results are means ± S.E. of four separate experiments, each performed in triplicate. *, statistically significant increase from untreated control cells.
Figure 2:
Effect of PDGF-stimulated putrescine
formation by vascular SMC. SMC were treated with PDGF (30 ng/ml) for 24
h in the presence of 50 µML-[C]ornithine (open
bars) or 1 mML-[
C]ornithine (closed
bars). Where indicated, L-NMA (10 mM) or DFMO (2
mM) was added to SMC treated with PDGF (30 ng/ml). Results are
means ± S.E. of three experiments, each performed in triplicate.
*, statistically significant increase from untreated control cells.
+, statistically significant effect of L-ornithine (1
mM) supplementation.
Treatment of vascular SMC with PDGF regulated the capacity of these cells to transport L-ornithine and other cationic amino acids. Time course studies demonstrated that PDGF (30 ng/ml) had a biphasic effect on cationic amino acid transport (Fig. 3). Initially, PDGF exposure inhibited the transport of cationic amino acids, but by 6 h of PDGF treatment a significant rise in cationic amino acid transport was observed, and this was further increased following 24 h of treatment (Fig. 3). The increase in basic amino acid uptake at 24 h was dependent on the concentration of PDGF (Fig. 4).
Figure 3:
Time course of PDGF (30 ng/ml) regulated
cationic amino acid transport by vascular SMC. Specific transport of 50
µML-[H]ornithine or L-[
H]arginine was measured for 45 s in
HEPES buffer following SMC preincubation with PDGF for the indicated
times. Results are means ± S.E. of three separate experiments,
each performed in triplicate. *, statistically significant effect of
PDGF treatment.
Figure 4:
Concentration-dependent increase in
cationic amino acid transport by PDGF in vascular SMC. SMC were treated
with PDGF (1-50 ng/ml) for 24 h, and then the specific transport
of 50 µML-[H]ornithine or L-[
H]arginine was measured for 45 s in
HEPES buffer. Results are means ± S.E. of three separate
experiments, each performed in triplicate. *, statistically significant
effect of PDGF.
In subsequent kinetic studies, saturable
uptake of radiolabeled L-arginine (0.005-10 mM)
was measured. As evident from the Eadie-Hofstee plots, uptake of L-arginine by SMC was biphasic, with two clearly separated
affinity states (Fig. 5). Similar results were obtained for L-ornithine uptake. A high affinity transporter having a
Michaelis constant (K) of 62 ± 13
µM and a maximum transport velocity (V
) of 1.24 ± 0.23 nmol/mg of protein/45
s was apparent. A second low affinity system was also demonstrated with
a K
of 2.48 ± 0.3 mM and a V
of 8.53 ± 1.41 nmol/mg of protein/45 s.
Pretreatment of vascular SMC with PDGF (30 ng/ml) for 24 h
significantly increased both the K
(107 ±
12 µM; p < 0.01) and V
(2.16 ± 0.06 nmol/mg of protein/45 s; p <
0.01) of the high affinity transporter (Fig. 5). Similarly, PDGF
pretreatment significantly elevated the V
(14.24
± 1.65 nmol/mg of protein/45 s; p < 0.05) of the low
affinity transporter but did not affect the K
(2.40 ± 0.4 mM) of this carrier (Fig. 5).
The increase in the V
of cationic amino acid
uptake by both carrier systems was completely abolished with
cycloheximide (5 µg/ml) (data not shown).
Figure 5:
Representative Eadie-Hofstee plot of
saturable L-arginine transport in vascular SMC. Specific
transport of L-[H]arginine
(0.005-10 mM) was measured for 45 s in control (open
circles) and PDGF (30 ng/ml for 24 h) pretreated (closed
circles) SMC. Transport velocity was plotted as a function of
velocity/L-arginine concentration (µM).
Regression analysis resolved transport into a high and low affinity
component. Similar findings were made in three separate experiments and
were also noted with L-ornithine
transport.
Incubation of SMC with the releasate from collagen-activated platelets for 24 h also stimulated the transport of cationic amino acids (Fig. 6). The addition of a PDGF neutralizing antibody to untreated SMC had no effect on cationic amino acid transport but prevented the increase in uptake evoked by the exogenous addition of PDGF (30 ng/ml), confirming the efficacy of the antibody (Fig. 6). Nonimmune IgG failed to modulate basic amino acid uptake in untreated cells and also had no effect on PDGF-induced increases in transport (Fig. 6). Treatment of the platelet releasate with a PDGF neutralizing antibody significantly reduced the ability of the platelet releasate to augment cationic amino acid uptake, although it did not completely reverse the stimulatory effect of the platelet releasate (Fig. 6). In contrast, nonimmune IgG did not modify the stimulatory effect of the releasate (Fig. 6).
Figure 6:
Effect of the platelet releasate on
cationic amino acid transport in vascular SMC. SMC were incubated with
the cell-free supernatant of 3 10
collagen (20
µg/ml) activated platelets for 24 h and specific transport of 50
µML-[
H]ornithine or L-[
H]arginine. Where indicated the
platelet releasate was incubated in the presence of a platelet-derived
growth factor neutralizing antibody (PDGF-Ab; 100 µg/ml) or
nonimmune IgG (100 µg/ml). The efficacy of the PDGF-Ab was tested
by examining its ability to inhibit the stimulation of cationic amino
acid transport evoked by PDGF (30 ng/ml for 24 h). Results are the
means ± S.E. of five separate experiments, each performed in
triplicate. *, statistically significant increase from untreated
control SMC. +, statistically significant inhibitory effect by
PDGF-Ab.
Reverse transcription coupled PCR identified cDNA encoding CAT-1 (630 bp) and CAT-2B (1150 bp) in untreated or PDGF-stimulated (30 ng/ml for 24 h) SMC, but in four out of five experiments failed to detect cDNA encoding CAT-2A (Fig. 7). In one experiment, an extremely faint CAT-2A band was detected (data not shown). No RT-PCR products were obtained with RNA samples in the absence of reverse transcriptase or when cDNA was omitted from the PCR reaction (data not shown). Ribonuclease protection assays demonstrated that PDGF treatment (3-50 ng/ml for 6 h) resulted in a concentration-dependent increase in the expression of both CAT-1 and CAT-2B; however, the PDGF-induced increase in CAT-2B message was much greater and was observed at lower PDGF concentrations (Fig. 8).
Figure 7: Ethidium-stained agarose electrophoresis gel showing PCR-amplified CAT cDNA from vascular SMC. Similar findings were made in four separate experiments.
Figure 8: Effect of PDGF on the expression of CAT mRNA by vascular SMC. This figure shows ribonuclease protection analysis of CAT-1, CAT-2B, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA from unstimulated (C) and PDGF-stimulated (1-50 ng/ml for 6 h) SMC. Similar findings were observed in three separate experiments.
The present study demonstrates that PDGF-induced SMC
mitogenesis is dependent on the transcellular transport of L-ornithine and its subsequent metabolism to polyamines. In
addition, we show that PDGF increases the transport of cationic amino
acids, such as L-ornithine, in vascular SMC by inducing the
expression of the genes encoding the system y transporter.
Recent studies indicate that SMC proliferation following PDGF treatment is dependent on polyamine synthesis(6) . Consistent with this, our data demonstrate that PDGF-stimulated DNA synthesis is associated with a prominent increase in the capacity of SMC to convert L-ornithine to putrescine. Furthermore, the selective ornithine decarboxylase inhibitor, DFMO(9) , blocks putrescine formation and inhibits thymidine incorporation into DNA. The exogenous addition of L-ornithine potentiates PDGF-induced putrescine formation and DNA synthesis in SMC. Both these effects of L-ornithine are reversed by treating SMC with a cationic amino acid transport inhibitor, L-NMA(13) , or by preventing the conversion of L-ornithine to putrescine with DFMO. These results suggest that the availability of L-ornithine is a limiting factor, and its metabolism to polyamines regulates the mitogenic response of PDGF in vascular SMC.
SMC can obtain L-ornithine from
extracellular sources via specific plasma membrane transport proteins
or from intracellular sources by protein degradation or by endogenous
synthesis. We have previously demonstrated that transport of
physiological levels of cationic amino acids (50-100
µM) by SMC is mediated by the
Na
-independent system y
carrier(13) . Our current study demonstrates that PDGF
can regulate the transport of cationic amino acid transport by this
carrier system in both a time- and concentration-dependent manner. Our
finding of a delayed increase in L-ornithine uptake
complements an earlier study, which demonstrated a similar
time-dependent increase in the activity of ornithine decarboxylase in
SMC following PDGF treatment(6) . These observations suggest
that PDGF-mediated increases in both L-ornithine uptake and
metabolism are coordinated to maximize the cellular capacity for
polyamine biosynthesis.
Kinetic experiments reveal the presence of
both a high (K
60 µM) and low
affinity (K
2.5 mM) carrier system
in SMC. Treatment of SMC with PDGF for 24 h increases the V
for both the high and low affinity
transporter, while the K
of only the high affinity
carrier is elevated. These kinetic data suggest that the PDGF-induced
increase in uptake observed at later time points results from the de novo expression of additional transport proteins. The
capacity of cycloheximide to abolish the PDGF-mediated increase in V
for both high and low affinity transport
systems is consistent with this notion. In contrast, the PDGF-induced
inhibition of uptake observed at early time points likely arises from a
PDGF-mediated decrease in affinity of the high affinity transporter.
We have found that vascular SMC express mRNA for CAT-1 and CAT-2B.
The co-expression of both CAT proteins is also observed in several
murine organs including stomach, skin, lung, and uterus, but contrasts
with the selective expression of CAT-2 in mouse
liver(29, 30, 31) . CAT-1 and CAT-2B possess
similar kinetic properties and are consistent with our kinetic data for
the high affinity carrier system, which, under physiologic conditions,
would mediate most of the transport of cationic amino acids by SMC.
Treatment of SMC with PDGF stimulates both CAT-1 and CAT-2B mRNA
expression. The concentration-dependent induction of CAT-2B message by
PDGF more closely parallels the PDGF-mediated elevation in cationic
amino acid transport; however, the relative contribution of CAT-1 and
CAT-2B to the overall activity of the system y carrier
is difficult to ascertain, owing to their similar uptake kinetics. The
recent finding that angiotensin II can also up-regulate CAT-1 and CAT-2
in vascular SMC suggests a general mechanism by which growth factors
can increase L-ornithine supply leading to elevated polyamine
production(32) . Thus, the capacity of vascular mitogens to
augment the transport of cationic amino acids via the induction of CAT
proteins provides not only the necessary amino acids required for the
synthesis of new proteins during cell growth, more importantly, it
plays an integral role in mediating the mitogenic response of vascular
SMC.
The uptake of cationic amino acids by vascular SMC is also
mediated by a low affinity carrier system whose transport kinetics
resemble CAT-2A. However, we could not consistently identify any CAT-2A
transcripts in SMC using a variety of primers based on the murine
sequence(19) . In addition, treatment of SMC with PDGF did not
induce CAT-2A expression. Whether this low affinity carrier system
represents a species variant of CAT-2A or a new transport protein is
not known. However, given its high K (
2.5
mM) for cationic amino acids, it is unlikely to play an
important physiological role. Our inability to detect CAT-2A message is
consistent with the suggestion by Closs et al.(18) that the alternate splice products of CAT-2 are
expressed in a tissue-specific manner.
Finally, the physiological
significance of our findings is suggested by the observation that the
releasate from collagen-activated platelets stimulates the transport of
cationic amino acids. This stimulatory effect of the platelet releasate
is largely mediated by the release of PDGF. However, other
platelet-derived factors are also likely to be involved since a
neutralizing antibody directed against PDGF can not fully reverse the
platelet-mediated increase in basic amino acid transport. The capacity
of activated platelets to induce the transport of L-ornithine
by vascular SMC may be of pathophysiological importance. Following
local injury of the vessel wall, platelets are recruited and
subsequently activated by interacting with subendothelial collagen,
releasing various mitogens, including PDGF, from their granules.
The ability of growth factors to augment L-ornithine uptake
would increase cellular polyamine production and thereby potentiate SMC
proliferation. Furthermore, our earlier observations of the capacity of
platelets to inhibit the expression of inducible nitric oxide synthase
in vascular SMC (28) would further promote intimal
proliferation by inhibiting the synthesis of the platelet inhibitory
and antiproliferative molecule, nitric oxide. Thus, the combined
ability of platelet-derived mitogens to up-regulate L-ornithine transport and metabolism and inhibit inducible
nitric oxide synthase expression may contribute to their SMC
proliferative actions at sites of vessel wall injury.
In conclusion, the present study demonstrates that PDGF-induced polyamine synthesis and SMC mitogenesis are dependent on the transcellular transport of L-ornithine. In addition, we show that PDGF can stimulate the transport of cationic amino acids in vascular SMC by inducing the expression of the genes for CAT-1 and CAT-2B. The capacity of PDGF to up-regulate the transport of L-ornithine may modulate its mitogenic effect by providing SMC with the necessary intracellular precursors for polyamine biosynthesis.