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 |
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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 |
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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 |
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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 ![]() |
RESULTS |
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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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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).
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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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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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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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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