Vol. 276, Issue 6, L1010-L1017, June 1999
Mechanisms regulating cAMP-mediated growth of bovine neonatal
pulmonary artery smooth muscle cells
Alexandra
Guldemeester1,
Kurt
R.
Stenmark2,
George H.
Brough3, and
Troy
Stevens3
2 Cardiovascular Pulmonary
Research Laboratory and Developmental Biology Laboratories, University
of Colorado Health Sciences Center, Denver, Colorado 80262;
1 Department of Pediatric Surgery,
Sophia Children's Hospital, 3015 GJ Rotterdam, The Netherlands; and
3 Department of Pharmacology,
University of South Alabama College of Medicine, Mobile, Alabama
36688
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ABSTRACT |
Neonatal pulmonary artery smooth muscle cells
(PASMCs) exhibit enhanced growth capacity and increased growth
responses to mitogenic stimuli compared with adult PASMCs. Because
intracellular signals mediating enhanced growth responses in neonatal
PASMCs are incompletely understood, we questioned whether
1)
Gq agonists increase cAMP content
and 2) increased cAMP is
proproliferative. Endothelin-1 and angiotensin II increased both cAMP
content and proliferation in neonatal but not in adult PASMCs.
Inhibition of protein kinase C and protein kinase A activity nearly
eliminated the endothelin-1- and angiotensin II-induced growth of
neonatal PASMCs. Moreover, cAMP increased proliferation in neonatal but not in adult cells. Protein kinase C-stimulated adenylyl cyclase was
expressed in both cell types, suggesting that insensitivity to
stimulation of cAMP in adult cells was not due to decreased enzyme
expression. Our data collectively indicate that protein kinase C
stimulation of cAMP is a critical signal mediating proliferation of
neonatal PASMCs that is absent in adult PASMCs and therefore may
contribute to the unique proproliferative phenotype of these neonatal cells.
adenosine 3',5'-cyclic monophosphate; lung; development; endothelin-1; angiotensin II; signal transduction
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INTRODUCTION |
ADAPTATION OF THE PULMONARY CIRCULATION to postnatal
life is a process that requires both growth and differentiation of
vascular wall cells. In smooth muscle cells (SMCs), there is a
transition from a fetal to a more adultlike phenotype (27). Several
studies (15, 26) have demonstrated that when the normal transition to
postnatal life is interrupted by hypoxia or increased pulmonary blood
flow, marked proliferative changes in pulmonary artery (PA) SMCs
(PASMCs) are observed that exceed those observed when adult animals are
exposed to these stimuli. Similarly, SMCs derived from neonatal
pulmonary arteries are less differentiated and exhibit enhanced growth
responses to mitogenic stimuli compared with the relatively
differentiated and quiescent SMCs derived from the adult pulmonary
artery (6, 30). Thus the increased growth capacity of neonatal PASMCs
likely contributes to both normal pulmonary vascular development and
the predisposition to develop exorbitant pulmonary vascular remodeling
in response to injury in the neonatal period.
Although it is generally accepted that neonatal PASMCs possess
increased growth responses to mitogenic stimuli, the unique intracellular signaling mechanism(s) that account for the enhanced growth responsiveness are incompletely understood. Ligands such as
insulin-like growth factor-I and platelet-derived growth factor are
coupled to tyrosine kinase signal transduction pathways that activate
extracellular signal-regulated kinases (ERKs) and potently increase
PASMC growth (2). Dempsey and colleagues (6, 8) demonstrated that
receptor tyrosine kinase-dependent agonists induced fourfold greater
increases in neonatal than in adult PASMC growth, suggesting that
ERK-dependent proliferation is developmentally controlled. It has
additionally been shown that constitutive and phorbol 12-myristate
13-acetate-sensitive protein kinase (PK) C activity is increased in
neonatal compared with adult PASMCs and that increased PKC activity
promotes neonatal PASMC growth and also synergistically promotes
ERK-dependent PASMC proliferation (6, 7). However, how PKC
synergistically interacts with ERK to enhance neonatal PASMC growth and
what accounts for enhanced PKC-dependent proliferation in neonatal
compared with adult SMCs is not clear at the present time.
Emerging data indicate that in some cell systems PKC may
synergistically promote ERK-dependent proliferation by elevating cAMP.
Faure and colleagues (11, 12) demonstrated that either Gq or
Gq
activation of PKC or direct
activation of PKC with phorbol esters stimulated ERK activity, and,
similarly, activation of Gs or
direct activation of adenylyl cyclase elevated cAMP and stimulated ERK
activity. Although these data implicate either Gq- or
Gs-coupled mechanisms in
regulation of ERK activity, they do not clearly demonstrate how
Gq-coupled agonists may elevate cAMP. However, recent elucidation of the molecular complexity of
adenylyl cyclases revealed type II adenylyl cyclase is activated by PKC
(32). These data suggest the possibility that a linkage between PKC and
ERK activation is PKC stimulation of type II adenylyl cyclase and
elevation of cAMP. Thus, as suggested by Faure and colleagues (11, 12),
Gq-coupled signal transduction
pathways activate PKC, which may promote adenylyl cyclase II synthesis of cAMP that, in turn, regulates ERK. It is equally clear that cAMP can
have opposite effects on ERK activity in other cell systems (11). Thus
second messenger regulation of ERK and proliferation may be unique in
phenotypically distinct cell types.
Because neonatal SMCs demonstrate unique PKC-associated growth
properties, it is possible that PKC regulation of cAMP may play an
important role in the increased growth responses in neonatal compared
with adult PASMCs. Therefore, the goal of the present study was to test
the hypothesis that Gq-coupled
agonists promote PKC-dependent stimulation of cAMP in neonatal PASMCs
and that such elevation of cAMP would be proproliferative. To test our hypothesis, we utilized two endogenous polypeptides, endothelin (ET)-1
and angiotensin II (ANG II), widely recognized as
Gq-coupled PKC agonists that
control SMC growth and differentiation (1, 9, 10, 13, 14, 16, 24, 25,
27). Both cAMP responses and indexes of proliferation were measured in
neonatal and adult PASMCs.
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METHODS |
Isolation and culture of neonatal and adult
PASMCs. SMCs were obtained from the main PAs of
neonatal (14-day-old) calves and adult (2-yr-old) cows. Neonatal and
adult PASMCs were considered matched because they were derived from the
middle media at the same vascular site with previously described
techniques (6-8). Briefly, main PAs were dissected from calves and
cows immediately after death and transported to the laboratory immersed
in MEM (pH 7.4) containing 200 U/ml of penicillin, 0.2 mg/ml of
streptomycin, and 5 mg/ml of amphotericin B at 25°C. The PAs were
opened, and the endothelium was scraped off. Explants of smooth muscle
tissue (2 × 3 mm) were dissected from the middle media of PA
strips. They were plated in petri dishes containing MEM with 10%
serum, 100 U/ml of penicillin, and 0.1 mg/ml of streptomycin. The
PASMCs at confluence exhibited characteristic "hill-and-valley"
(adult) and "swirllike" (neonatal) morphologies by phase-contrast
microscopy and stained in a homogeneous fibrillar pattern with smooth
muscle-specific monoclonal anti--smooth muscle actin antibody
(Sigma, St. Louis, MO) (6-8). Cell cultures were maintained in MEM
(pH 7.4) containing 1%
L-glutamine, 200 U/ml of
penicillin, 0.2 mg/ml of streptomycin, and 0.5% MEM-nonessential amino
acid solution (all from Sigma) with 10% bovine calf serum (BCS;
Hyclone Laboratories, Logan, UT) and incubated in a humidified
atmosphere with 5% CO2 at
37°C. The medium was changed biweekly. To ensure that any
differences seen between the cell populations were due to intrinsic
differences and not induced in vitro, we controlled identical sites of
harvest, time in culture, passage number, and growth conditions of the neonatal and adult PASMCs. The cells were studied between primary culture and third passage. Cells were grown to confluence in T75 flasks
in the presence of 10% BCS, removed from the tissue culture flasks by
trypsinization (0.2 g/l of trypsin-0.5 g/l of EDTA; Sigma), and then
seeded at equal density into 24-well tissue culture plates (50 × 103 cells/well). Cells were grown
to confluence in the presence of 10% serum in 2-3 days and
incubated for 72 h in serum-deprived medium (0.1% BCS) to achieve a
quiescent state.
Measurement of [3H]thymidine
incorporation into DNA.
DNA synthesis was measured as previously described (6-8). For
these experiments, neonatal and adult PASMCs were grown to confluence,
and a quiescent state was achieved after 72 h in 0.1% BCS-MEM.
[3H]thymidine (0.5 µCi/well; ICN Biochemicals, Irvine, CA) was added together with ET-1,
ANG II, forskolin, or 8-bromo-cAMP (Sigma) for 24 h. In studies of
[3H]thymidine
incorporation during PKC or cAMP blockade, the cells were pretreated
with the specific PKC inhibitors chelerythrine chloride and Ro 31-8220 or the cAMP antagonist Rp diasteromer of adenosine 3',5'-cyclic monophosphothioate
(Rp-cAMPS; all Alexis, San Diego, CA) for 15 min, followed by application of
[3H]thymidine (0.5 µCi/well) together with ET-1, ANG II, forskolin, or 8-bromo-cAMP for
24 h. Cell counts were obtained at the end of the incubation with a
hemocytometer. After the cells were washed with phosphate-buffered
saline (PBS) and 0.2 M perchloric acid (0.5 ml/well) was added for
2-3 min, the cells were again washed with PBS (1 ml/well), and
then 1.0% SDS-0.01 N NaOH (0.3 ml/well) was added. The contents of
each well were added to 4 ml of Ecoscint H scintillation cocktail
(National Diagnostics, Atlanta, GA), and the radioactivity was measured
with a Beckman LS 7500 beta-scintillation counter (Irvine, CA).
Incorporation of
[3H]thymidine into DNA
is expressed as counts per minute (cpm) per cell.
Measurement of change in cell number.
Cells were trypsinized for 10 min, gently triturated four times after
the addition of an equal volume of MEM-10% serum, and counted with a
standard hemocytometer.
Measurement of cAMP accumulation. cAMP
measurements were made with confluent, quiescent neonatal and adult
PASMCs grown to confluence in 24-well plates (plated at 50 × 103 cells/well) with a standard
radioimmunoassay (Biomedical Technologies, Stoughton, MA). Studies were
conducted with MEM at pH 7.35-7.45. In studies of cAMP
accumulation, either vehicle control, ET-1, or ANG II was added to the
cells, and the cells were incubated at 37°C for 90 min. In selected
experiments, the cAMP signal was amplified with the -adrenergic
agonist isoproterenol (Sigma) along with the vehicle control, ET-1, or
ANG II. In all studies, the solutions contained the phosphodiesterase
inhibitor 3-isobutyl-1-methylxanthine (IBMX; Sigma) to inhibit cAMP
breakdown. In studies of cAMP accumulation during PKC blockade, the
cells were pretreated with the specific PKC inhibitors chelerythrine
chloride or Ro 31-8220 for 15 min, followed by application of the
vehicle control, ET-1, or ANG II. After incubation, the cells were
washed with PBS, and the reactions were stopped with 1 M NaOH and then
neutralized to pH 
7.0 with 1 M HCl. The solutions were acetylated,
the tubes were centrifuged, and the supernatant was decanted.
Radioactivity of the precipitate was counted, and sample cAMP was
calculated from a standard curve. cAMP was standardized to cell counts
obtained from untreated wells at the end of the incubation.
RT-PCR detection of adenylyl cyclase.
Expression of PKC-stimulated adenylyl cyclase (type II) was tested
generally as previously described (4). Briefly, total RNA was extracted
from cell monolayers with Qiagen RNeasy (Qiagen, Santa Clarita, CA) and
subjected to first-strand synthesis with reverse transcriptase (GIBCO
BRL) and oligo(dT) primer after treatment of RNA with DNase I (GIBCO BRL). Degenerate primers directed to the highly conserved
C2A region of adenylyl cyclase
were used for PCR amplification (28). PCR products were ligated into a
cloning vector (TOPO TA cloning kit, Invitrogen, San Diego, CA) and
transformed into competent cells. After PCR screening of clones for
proper inserts, bacterial cultures were made and grown for 16-18
h. Plasmids were purified by QIAprep Spin Plasmid kit (Qiagen).
Sequencing was performed by automated fluorescence sequencing (ABI370A
DNA Sequencer).
Lung fixation. Neonatal calves were
killed by an intravenous overdose of barbiturate and exanguinated
before removal of the lungs. The superior lobe of the left lung was
fixed in Formalin and cut, and lung blocks were embedded in paraffin.
Sections of paraffin-embedded tissue were cut with a microtome at 4 µm. Before the slides were stained, the paraffin was removed from the
slides with xylene, and the tissue was rehydraded in a graded alcohol series.
Immunohistochemistry. Immunostaining
was generally as previously described (4, 17). To test for adenylyl
cyclase expression, the slides were incubated in 0.3%
H2O2
in methanol for 30 min to decrease endogenous peroxidase activity. The
slides were incubated with blocking solution (1% BSA and 0.05% Triton
X-100 in PBS) to reduce nonspecific binding of antibodies. Primary
antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) for type II
adenylyl cyclase were diluted 1:250 in PBS with 0.1% BSA and 0.05%
Triton X-100 and incubated overnight at room temperature. After the
slides were washed, biotinylated goat anti-rabbit IgG antibody diluted 1:250 in PBS with 0.05% Tween was added for 2 h. The slides were washed and incubated in an avidin-biotin-horseradish peroxidase complex
(Vectastain ABC Kit, Vector Laboratories, Burlingame, CA) diluted 1:250
in PBS with 0.05% Tween for 1.5 h. The slides were again washed and
developed in 5 mg of diaminobenzidine, 10 ml of 50 mM Tris, pH 7.4, and
10 µl of 30%
H2O2
for 1 min, rinsed with tap water, and counterstained briefly with
hematoxylin before dehydration and mounting. In control experiments,
blocking peptide (Santa Cruz Biotechnology) was diluted 1:250 and
coincubated with the type II adenylyl cyclase polyclonal antibody.
The antibody for type II adenylyl cyclase was generated against the
sequence KTYFVNTEMSRSLSQSNVAS of the type II enzyme. Specificity of the
antibody for type II adenylyl cyclase has been confirmed by Santa Cruz
Biotechnology with Western blotting and immunocytochemistry.
Statistical analysis. Data are
reported as means ± SE. One-way ANOVA with multiple comparisons was
used to compare means between groups. A
P value < 0.05 was used to indicate significance.
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RESULTS |
Stimulation of cAMP. To address
whether ET-1 and ANG II could increase proliferation in neonatal PASMCs
through a cAMP-mediated pathway, we first measured cAMP levels in
PASMCs in the presence of the phosphodiesterase inhibitor IBMX (500 µM) and in response to ET-1 and ANG II. Baseline cAMP was higher in
neonatal PASMCs than in adult cells (Fig.
1A).
Both ET-1 (10 nM) and ANG II (10 nM) stimulated cAMP synthesis (12-
and 4-fold, respectively; P < 0.05;
n = 6 cells) in neonatal PASMCs over
90 min but did not change cAMP levels in adult cells (Fig.
1A). Similar results were observed
in response to ET-1 and ANG II in the presence of isoproterenol (25 µM) and IBMX over a 5-min time course (data not shown).
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Fig. 1.
Endothelin (ET)-1 and ANG II activate protein kinase (PK) C and
increase cAMP in neonatal pulmonary artery (PA) smooth muscle cells
(SMCs). Cells were plated at 50,000 cells/well and studied in a
confluent and quiescent state. After application of vehicle or
treatment conditions, cAMP was measured with radioimmunoassay and
standardized to cell counts. Values are means ± SE.
A: baseline cAMP in presence of IBMX
was higher in neonatal than in adult PASMCs. ET-1 (10 nM) and ANG II
(10 nM) increased baseline cAMP over a 90-min time course in neonatal
but not in adult cells (n = 6/group). Significantly different (P < 0.05) from: * control PASMCs; ** neonatal PASMCs.
B: pretreatment with (+) chelerythrine
(1 µM) reduced baseline cAMP and eliminated ET-1- and ANG II-induced
rise in cAMP in neonatal PASMCs (n = 6/group).  , Without. * Significantly different from
without chelerythrine, P < 0.05.
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We investigated the possibility that ET-1 and ANG II increased cAMP in
neonatal PASMCs by PKC-mediated stimulation of adenylyl cyclase.
Pretreatment of these cells with the PKC inhibitor chelerythrine (1 µM) reduced baseline cAMP (30%) and eliminated ET-1 and ANG II
stimulation of cAMP (Fig. 1B).
Similar results were obtained with the specific PKC blocker Ro 31-8220 (5 µM; data not shown). These data are consistent with the idea that
basal cAMP levels and elevation of cAMP after application of ET-1 and
ANG II are regulated through PKC stimulation of adenylyl cyclase.
Expression of type II (PKC-stimulated) adenylyl
cyclase in vitro and in vivo. The activity of type II
adenylyl cyclase is stimulated by PKC (32). We next sought to identify
whether the type II enzyme is expressed in neonatal PASMCs using RT-PCR
cloning. Sequence analysis of a 261-nucleotide product revealed 94 and 90% homology between the bovine product and the respective human and
rat species at the nucleotide level. Deduced amino acid alignments demonstrated 97 and 95% homology between the presently cloned bovine
and respective human and rat sequences (Table
1).
Although type II adenylyl cyclase is expressed in cells in culture,
isolation and culture per se may induce phenotypic changes in the
population of PASMCs. Thus we determined whether PKC-stimulated adenylyl cyclase was evident in neonatal and adult PASMCs in vivo using
established immunohistochemical techniques. A recently developed adenylyl cyclase type II-specific polyclonal antibody was utilized. We
found expression of type II adenylyl cyclase throughout the pulmonary
vasculature. Especially important is detection of the type II
PKC-stimulated adenylyl cyclase in the medial layer at the site where
excessive proliferation of PASMCs in response to mitogenic stimuli
occurs (Fig. 2). Immunoreactivity was
eliminated by coincubation of the PKC-stimulated adenylyl cyclase type
II antibody with a blocking peptide, suggesting antibody specificity to
type II adenylyl cyclase.
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Fig. 2.
PKC-stimulated adenylyl cyclase (type II) is expressed in pulmonary
arteries and arterioles of neonatal and adult animals. Diaminobenzadine
staining (brown) was used to determine expression of type II adenylyl
cyclase in intact pulmonary circulation. Antibody specificity was
tested by blocking diaminobenzadine staining with peptide inhibitors
against primary antibody (A and
C). Medial sections of small
(B) and large
(D) neonatal pulmonary arteries
stained positive with adenylyl cyclase type II-specific polyclonal
antibody. Most SMCs in medial layer of small and large pulmonary
arteries stained brown (B and
D, arrowheads), demonstrating
expression of adenylyl cyclase type II. Data with pulmonary vessels
from adult animals are not shown. Hematoxylin was used to counterstain
cell nuclei (purple).
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SMC proliferation. To assess the
contribution of cAMP to growth in neonatal and adult PASMCs, we
measured the effects of the direct adenylyl cyclase agonist forskolin
(10 µM) and the cAMP analog 8-bromo-cAMP (1 µM) on
[3H]thymidine
incorporation. Basal
[3H]thymidine
incorporation was threefold higher in neonatal than in adult cells.
Forskolin (10 µM) and 8-bromo-cAMP (1 µM) induced a twofold
increase in
[3H]thymidine
incorporation in neonatal PASMCs but did not affect [3H]thymidine
incorporation in adult PASMCs (P < 0.05; n = 4 cells; Fig.
3). Cell counts after forskolin and
8-bromo-cAMP application were higher in neonatal but not in adult
PASMCs (data not shown), consistent with a proproliferative effect of
cAMP in these neonatal cells (30).
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Fig. 3.
Elevated cAMP promotes growth in neonatal PASMCs. Cells were plated at
50,000 cells/well and studied in a confluent and quiescent state.
After application of treatment conditions, incorporation of
[3H]thymidine into DNA
was measured. cpm, Counts/min. Values are means ± SE;
n = 4 cells/group. Basal
[3H]thymidine
incorporation in neonatal PASMC was threefold higher than in adult
cells. Forskolin (10 µM) and 8-bromo-cAMP (1 µM) increased
[3H]thymidine
incorporation after 24 h in neonatal but not in adult cells.
Significantly different (P < 0.05) from: * control PASMCs; ** neonatal PASMCs.
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Our next studies investigated whether neonatal PASMCs exhibit greater
growth responses than adult cells to the
Gq agonists ET-1 (10 nM) and
ANG II (10 nM). ET-1 and ANG II increased
[3H]thymidine
incorporation three- and twofold, respectively, in neonatal PASMCs but
did not increase
[3H]thymidine
incorporation in adult PASMCs (P < 0.05; n = 4 cells; Fig.
4). Cell counts after ET-1 and ANG II
application were also higher in neonatal but not in adult PASMCs (data
not shown).
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Fig. 4.
ET-1 and ANG II activate PKC and stimulate growth of neonatal PASMCs.
Cells were plated at 50,000 cells/well and studied in a confluent and
quiescent state. After application of treatment conditions,
incorporation of
[3H]thymidine into DNA
was measured. Values are means ± SE;
n = 4 cells/group.
A: ET-1 (10 nM) and ANG II
(10 nM) increased
[3H]thymidine
incorporation after 24 h in neonatal but not in adult cells.
Significantly different (P < 0.05)
from: * control PASMCs; ** neonatal PASMCs.
B: pretreatment with chelerythrine (1 µM) inhibited ET-1- and ANG II-stimulated neonatal PASMC
proliferation. * Significantly different from without
chelerythrine, P < 0.05.
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We investigated the role of PKC in the proliferative response to ET-1
and ANG II in neonatal PASMCs using chelerythrine. Figure 4B shows that PKC inhibition
attenuated the basal and ET-1- and ANG II-mediated increase in
[3H]thymidine
incorporation (6, 75, and 75%, respectively;
P < 0.05; n = 4 cells). Identical results were
achieved with the PKC blocker Ro 31-8220 (5 µM; data not shown). A
previous report from our laboratory (30) has shown that PKC inhibitors
at concentrations presently reported do not cause significant cell
death, confirming that inhibition of PKC decreased proliferation rather
than induced apoptosis or necrosis. We next tested the role of adenylyl
cyclase and cAMP in increased proliferation by blocking cAMP-dependent protein kinase activity with Rp-cAMPS
(1 mM). Rp-cAMPS attenuated basal and
ET-1- and ANG II-mediated increases in
[3H]thymidine
incorporation (6, 85, and 78%, respectively;
P < 0.05; n = 4 cells; Fig.
5), confirming a proproliferative action of cAMP in neonatal PASMCs. Altogether, these data suggest that
stimulation of proliferation in quiescent neonatal PASMCs is at least
partly regulated through PKC stimulation of adenylyl cyclase and cAMP.
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Fig. 5.
Inhibition of PKA prevents ET-1 and ANG II stimulation of neonatal
PASMC growth. After pretreatment with
Rp diasteromer of adenosine
3',5'-cyclic monophosphothioate
(Rp-cAMPS; 1 mM) and application of
ET-1 or ANG II, incorporation of
[3H]thymidine into DNA
was measured. Values are means ± SE;
n = 4 cells/group. PKA blockade
attenuated basal and ET-1- and ANG II-induced increase in
[3H]thymidine
incorporation in neonatal PASMCs. * Significantly different from
neonatal PASMCs, P < 0.05.
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DISCUSSION |
Vascular SMCs derived from neonatal PAs exhibit enhanced growth
capacities to growth-promoting stimuli compared with SMCs derived from
adult pulmonary arteries (6, 30). The reason for this unique phenomenon
is unclear, although enhanced growth capacity may contribute to normal
adaptive mechanisms after birth as well as the need for continued
pulmonary vascular growth. Recent evidence (2, 6-8) indicated that
compared with adult PASMCs, neonatal PASMCs exhibit enhanced growth
responses to activation of ERK and PKC. Moreover, stimulation of ERK
occurred after elevation of cAMP, suggesting that PKC stimulation of
cAMP may be a critical link to ERK-dependent proliferation (2, 6, 8,
11, 12, 22). However, it was unclear whether activation of PKC
influences PASMC cAMP content and whether cAMP is proproliferative in
PASMCs. Novel findings from our study are that
1)
Gq agonists ET-1 and ANG II
elevate neonatal but not adult PASMC cAMP,
2) both neonatal and adult PASMCs
express a PKC-stimulated adenylyl cyclase, and 3) ET-1, ANG II, and direct
elevation of cAMP is proproliferative in neonatal but not in adult
PASMCs. These data suggest that PKC stimulation of cAMP is a critical
signal mediating proliferation of neonatal PASMCs that is absent in
adult PASMCs and therefore may contribute to the unique
proproliferative phenotype of neonatal PASMCs.
Our initial studies sought to determine whether
Gq agonists ET-1 and ANG II
promote cAMP synthesis. Activation of PKC in diverse cell types,
including bronchial SMCs, increases cAMP content (22). Both ET-1 and
ANG II increased cAMP content in PASMCs over a 90-min time course, and
inhibition of PKC prevented the ET-1- and ANG II-induced rise in cAMP.
Interestingly, neither Gq agonist
tested elevated cAMP content in adult PASMCs. Thus these data are the first to demonstrate that PKC stimulation of cAMP is developmentally controlled.
Recent elucidation of multiple adenylyl cyclase species revealed that
certain isoforms (e.g., type II) are stimulated by PKC (32), providing
a putative mechanism through which PKC may increase cAMP content. Our
next studies therefore determined whether PKC-stimulated adenylyl
cyclase was selectively expressed in neonatal PASMCs. We tested
expression of the type II isoform by RT-PCR cloning using
sequence-specific oligonucleotide primers. Sequence analysis revealed
that type II adenylyl cyclase is expressed in both neonatal and adult
PASMCs. To confirm that expression of this enzyme was not an artifact
of cell culture per se, immunostaining was performed on sections from
intact neonatal and adult bovine lungs. Positive staining was observed
in the medial layers of large and small vessels from animals of both
developmental stages. Thus these data indicate that the expression of
type II adenylyl cyclase is not developmentally controlled and does not
account for the distinct ET-1 and ANG II responses in neonatal versus
adult PASMCs.
Although PKC-stimulated adenylyl cyclase is expressed in both neonatal
and adult cells, our data indicated that PKC only stimulated the type
II enzyme in neonatal PASMCs, supporting the idea that mechanisms
controlling adenylyl cyclase activation are developmentally regulated.
Dempsey et al. (6) previously demonstrated that relative to adult
cells, neonatal PASMCs exhibit increased PKC activity under basal
conditions and increased sensitivity to the direct PKC activator
phorbol 12-myristate 13-acetate. It is therefore reasonable that
increased PKC activity in neonatal PASMCs stimulates type II adenylyl
cyclase, whereas lower PKC activity in adult cells does not stimulate
type II adenylyl cyclase. Multiple isoforms of PKC are present in
neonatal PASMCs, but the -isozyme has been implicated in increased
growth responses (30). Interestingly, the -isozyme of PKC activates
type II adenylyl cyclase in Sf9 cells (34). Future studies will be
required to directly test the nature of PKC stimulation of adenylyl
cyclase activity in neonatal PASMCs, e.g., which PKC isoforms account
for increased whole cellular PKC activity and activation of type II
adenylyl cyclase.
Our next studies were designed to address whether a link exists between
PKC stimulation of cAMP and neonatal PASMC proliferation by determining
whether 1) elevated cAMP is
proproliferative, 2) PKC activation
is proproliferative, and 3) PKC
stimulation of proliferation depends on cAMP. The role of cAMP on SMC
proliferation is controversial. Previous reports suggested that cAMP
may have either a negative or positive influence on proliferation (23), with the effect of cAMP depending on cell type (23), state of cell
differentiation (3), and stage of cell cycle (21). Neonatal and adult
PASMCs were "growth arrested" to mimic the in vivo environment. Although previous studies (6, 30) showed that neonatal PASMCs exhibit
enhanced growth capabilities, our present studies demonstrated that
these cells also exhibit higher basal cAMP levels, consistent with the
possibility that cAMP may function as a positive stimulus for
proliferation. We found that two agents that increase cAMP (8-bromo-cAMP and forskolin) also stimulate
[3H]thymidine
incorporation and cell proliferation in neonatal but not in adult
PASMCs. Furthermore, direct inhibition of the cAMP-dependent PK lowered
basal [3H]thymidine
incorporation in neonatal PASMCs. These data suggest that cAMP is
proproliferative in neonatal PASMCs and that the action of cAMP-induced
growth is developmentally regulated as recently suggested in Schwann
cells (31).
We next evaluated the influence of PKC on growth in neonatal PASMCs.
PKC activity is increased in neonatal versus adult PASMCs, and ET-1 and
ANG II activate PKC. Furthermore, activation of PKC is generally found
to stimulate proliferation (6, 16, 20, 25, 34). In our present studies,
inhibition of PKC with chelerythrine and Ro 31-8220 decreased basal and
ET-1- and ANG II-stimulated [3H]thymidine
incorporation, suggesting that increased growth in neonatal PASMCs
depends at least partly on PKC activity. However, PKC inhibitors did
not influence proliferation in adult PASMCs. Both ET-1 and ANG II are
generally believed to stimulate growth in adult SMCs derived from the
systemic circulation (1, 5, 10, 27). Indirect evidence for the
involvement of ET-1 and ANG II in medial thickening of pulmonary
arteries has also been shown in adult rats (18, 33), but the direct
effects of the polypeptides on PASMC proliferation are less clear. For
example, ET-1 was previously reported (16) to increase growth in adult swine PASMCs in the presence of 0.5% serum, whereas in the present study, ET-1 and ANG II were not proproliferative in adult bovine PASMCs
in the presence of 0.1% serum. The reason for this discrepancy is
unclear, although it is possible that these agents act as comitogenic stimuli, requiring other growth factors to stimulate proliferation. Independent support for this idea comes from the work of Morrell and
Stenmark (19), who observed that ANG II stimulated proliferation of
adult rat PASMCs only when the cells were primed by preincubation with
10% serum but not under serum-deprived conditions (0.1%). Thus our
data are consistent with the idea that ET-1 and ANG II alone are
insufficient to promote proliferation in adult PASMCs and suggest that
PASMCs possess a developmentally regulated sensitivity to these
vasoconstrictors (29).
Our final series of experiments tested whether inhibition of PKA blocks
ET-1- and ANG-II-stimulated increase in neonatal PASMC proliferation.
Indeed, the PKA inhibitor Rp-cAMPS
prevented Gq activation from
stimulating neonatal PASMC proliferation but did not
affect proliferation of adult PASMCs. Our data therefore
demonstrate that ET-1 and ANG-II stimulate PKC-dependent production of
cAMP that is proproliferative in neonatal PASMCs; inhibitors of either PKC or PKA prevent this stimulation of proliferation.
In summary, ET-1 and ANG-II activation of
Gq activates PKC, which increases
cAMP and promotes proliferation of neonatal PASMCs. In contrast, ET-1
and ANG II activation of Gq
neither increases cAMP nor promotes proliferation of adult PASMCs. The
explanation for this apparent developmental distinction is not yet
fully determined but is not due to altered expression of PKC-stimulated
(type II) adenylyl cyclase. Based on earlier work from our
laboratory (6, 8), a likely explanation is that increased
constitutive PKC activity and enhanced PKC responsiveness to activation
accounts for PKC stimulation of cAMP in neonatal versus adult PASMCs.
Now that a key link between PKC and cAMP production has been
established in neonatal PASMCs, future studies may address the
regulation of ERK-dependent proliferation by cAMP.
| |
ACKNOWLEDGEMENTS |
We thank Sandi Walchak for excellent technical assistance, Stephen
Hofmeister for figure preparation, and Drs. J. V. Weil and M. N. Gillespie for valuable discussion of the manuscript.
| |
FOOTNOTES |
This work was supported by grants from The Ter Meulen Fund, Royal
Netherlands Academy of Arts and Sciences (Amsterdam, The Netherlands)
(to H. A. Guldemeester); Parker B. Francis Fellowship (to T. Stevens);
National Heart, Lung, and Blood Institute (NHLBI) Specialized Center of
Research Grant HL-57144; NHLBI Program Project Grant HL-14985 (to K. R. Stenmark); and NHLBI Grants HL-56050 and HL-60024 (to T. Stevens).
Address for reprint requests and other correspondence: T. Stevens,
Dept. of Pharmacology, Univ. of South Alabama College of Medicine, MSB
3130, Mobile, AL 36688-0002 (E-mail:
tstevens{at}jaguar1.usouthal.edu).
Received 4 December 1997; accepted in final form 16 February 1999.
| |
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ABSTRACT
INTRODUCTION
