Departments of 1 Pathology and 2 Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2650
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ABSTRACT |
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We examined the hypothesis that the
potent vasoconstrictor endothelin (ET)-1 regulates both its own
production and production of the vasodilator prostaglandins
PGE2 and prostacyclin in sheep peripheral lung vascular
smooth muscle cells (PLVSMC). Confluent layers of PLVSMC were exposed
to 10 nM ET-1; expression of the prepro (pp)-ET-1, cyclooxygenase
(COX)-1, and COX-2 genes was examined by RT-PCR and Western
analysis. Intracellular levels of ET-1 were measured by ELISA
with and without addition of the protein synthesis inhibitor brefeldin
A (50 µg/ml). Prostaglandin levels were measured by gas
chromatography-mass spectrometry. Through use of ETA and
ETB antagonists (BQ-610 and BQ-788, respectively), the
contribution of the ET receptors to COX-1 and -2 expression and ppET-1
gene expression was examined. The contribution of phosphorylated p38
and p44/42 MAPK on COX-1 and COX-2 expression was also examined with
MAPK inhibitors (p38, SB-203580 and p44/42, PD-98056). ET-1 resulted in
transient increases in ppET-1, COX-1, and COX-2 gene and protein
expression and release of 6-keto-PGF1 and
PGE2 (P < 0.05). Both internalization of
ET-1 and synthesis of new peptide contributed to an increase in
intracellular ET-1 (P < 0.05). Although increased
ppET-1 was regulated by both ETA and ETB, COX-2
expression was upregulated only by ETA; COX-1 expression was unaffected by either antagonist. ET-1 treatment resulted in transient phosphorylation of p38 and p44/42 MAPK; inhibitors of these
MAPKs suppressed expression of COX-2 but not COX-1. Our data indicate
that local production of ET-1 regulates COX-2 by activation of the
ETA receptor and phosphorylation of p38 and p44/42 MAPK in PLVSMC.
prostacyclin; prostaglandin E2; endothelin receptors
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INTRODUCTION |
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A MIXED POPULATION of
muscular, partially muscular, and nonmuscular arteries (<150 µm in
diameter) exist in the intra-acinar region of the lung. Pericytes and
intermediate cells (precursor smooth muscle cells as indicated by their
content of smooth muscle -actin and myosin filaments) have been
demonstrated in the walls of the non- and partially muscular arteries,
respectively, and have been ascribed a role in local homeostasis
(22, 29, 30). Local production of vasodilator and
vasoconstrictor prostaglandins and the potent vasoconstrictor
endothelin-1 (ET)-1 have been suggested to contribute to the local
homeostasis in the pulmonary vasculature, although little is known of
the metabolic activity of the smooth muscle-like cells and even less
about the interplay between these vasoactive agents.
ET-1 is produced by cells in many tissues, including endothelium and vascular smooth muscle cells (15, 24, 36). Of the three isoforms of ET (ET-1, ET-2, and ET-2), ET-1 is the major form produced in the lung (21), where it has been shown to act both as a potent vasoconstrictor and also to invoke early vasodilation (24). The intracellular effects of ET-1 are triggered by the G protein-coupled ET receptors ETA and ETB. In a previous study, we demonstrated regional variability in expression of the prepro (pp)-ET-1 gene in the normal sheep pulmonary artery, in that ppET-1 transcripts are lower in the midregion pulmonary artery than in the main pulmonary artery and peripheral lung (12). Such differences in ppET-1 gene expression have also been found in the lungs of rats (5, 6). Although it is doubtless that the endothelial cells contribute to the variations in local ET-1 gene expression, whether peripheral lung vascular smooth muscle cells (PLVSMC) also contribute to local synthesis of ET-1 is not known.
Prostaglandins, including prostaglandin E2 (PGE2) and prostacyclin (PGI2), are known to modulate various lung functions including pulmonary vascular tone (19). Synthesis of prostanoids is orchestrated in a cell-specific fashion (14). Cyclooxygenases (COX) are the rate-limiting enzymes in the biochemical processing of arachidonic acid to vasoactive prostanoids (43). At least two distinct COX enzymes are recognized (31, 41). COX-1 is generally thought to be expressed constitutively and to produce prostaglandins that modulate normal vascular tone. COX-2 is the inducible form of the enzyme and has been linked to many proinflammatory stimuli (13, 19, 32). Stimulation of COX-2 expression requires activation of MAPK and expression of certain transcription factors (17, 38). Furthermore, the particular MAPK involved appears to vary from one cell type to another (16, 20). Whether PLVSMC synthesize prostaglandins and whether regulation of COX and prostaglandin synthesis and release are regulated by exogenous ET-1 is not certain.
The present study examines the metabolic activity of PLVSMC isolated
and cultured from the normal sheep lung. In particular, we examine
basal expression of COX-1 and COX-2 and prostaglandin production, as
well as basal expression of the ppET-1 gene and their responses to
exogenous ET-1. Furthermore, using specific MAPK inhibitors, we examine
whether these changes are mediated through phosphorylation of the p38
MAPK and p42/44 (ERK), and through use of ET receptor antagonists, we
also examine the role of the ETA and ETB
receptors to induction of these genes. Our data demonstrate that
1) PLVSMC express COX-1, COX-2, and ppET-1 at baseline and
release both PGE2 and 6-keto-PGF1
(prostacyclin), and 2) stimulation with ET-1 upregulates
each of these genes and prostaglandin production. Stimulation of COX-2,
but not COX-1, by ET-1 is dependent on phosphorylation of MAPKs and
follows activation of the ETA receptor.
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METHODS |
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Isolation of PLVSMC. PLVSMC were isolated using a modification of the iron oxide magnetic separation technique (22). Briefly, the lungs were removed intact from normal sheep, and the pulmonary artery and bronchus supplying the upper segment of the right lower lobe were cannulated. The artery was flushed with phosphate-buffered saline (PBS) while the segment was "ventilated" with room air by way of a 25-ml syringe inserted into the cannula in the right lower lobe. When the PBS exiting the pulmonary vein was clear, the segmental artery was injected with a suspension of 0.5% iron oxide particles in 0.5% low-melting-point agarose (Omnipure; VWR, Atlanta, GA) in culture medium I [Dulbecco's modified Eagle's medium (DMEM); GIBCO Laboratories, Grant, NY] supplemented with 100 U/ml penicillin, 100 U/ml streptomycin, and 25 mM HEPES at 45°C (GIBCO). The preparation was then transferred to a beaker containing ice-cold PBS for 10 min to set the agar. The segment was dissected from the rest of the lobe, the pleural surface was removed, and a strip of lung <1 mm in thickness containing the filled black microvessels was taken. Using a dissecting microscope, we dissected and discarded areas containing vessels >100 µm, and the remaining lung was minced with fine scissors, suspended in culture medium I and placed in a magnetic separator (Promega, Madison, WI). The mince was rinsed, digested with 80 U/ml collagenase (Worthington Biochemicals, Lakewood, NJ) for 30 min at 37°C, and sheared by several strokes through an 18-gauge needle. The PLVSMC were then isolated magnetically and suspended at low density in culture medium II (culture medium I supplemented with 20% FBS) in P100 dishes. Clones of PLVSMC were trypsinized and passed into six-well plates. All experiments were carried out on cells between passages 3 and 6.
Characterization of PLVSMC.
PLVSMC were identified by their stellate shape as seen via
phase-contrast microscopy. Their smooth muscle nature was confirmed using antibodies to smooth -actin and smooth muscle myosin
(Sigma-Aldrich, St. Louis, MO) and immunocytochemical techniques as
previously described (39).
Experimental protocols. All experiments were carried out in DMEM containing 0.1% bovine serum albumin (Sigma). Experiments were initiated following an overnight incubation of the cells in that medium. A dose of 10 nM ET-1 (Peptides International, Louisville, KY) was used in all experiments. This is a dose that we have found causes a 50% increase in intracellular ET-1 in inner medial pulmonary artery smooth muscle (L2) cells (40) and in PLVSMC causes peak expression of the COX-2 gene (data not shown). In experiments in which we utilized ET-1 receptor antagonists, cells were pretreated with either 0.5 µM BQ-610 (ETA receptor antagonist) and/or 25 µM BQ-788 (ETB receptor antagonist) (Peptide Institute, Osaka, Japan) for 2 h before treatment with ET-1. The doses for the receptor antagonists correspond to the ED50 for downregulation of ET-1 internalization in L2 cells from sheep main pulmonary artery. In experiments to examine the MAPK activity, we pretreated PLVSMC with the MAPK inhibitors SB-203580 (10 µM, p38) and/or PD-98059 (25 µM, ERK; Sigma) for 1 h (16, 20). To determine whether PLVSMC synthesize ET-1, we measured intracellular levels of ET-1 with and without 15-min pretreatment with the protein synthesis inhibitor brefeldin A (BFA, 50 µg/ml; Sigma) (2, 37) before a 4-h incubation with exogenous ET-1 (10 nM).
Western blot analysis. The temporal sequences of ET-1-stimulated COX-1 and COX-2 expression and phosphorylation of the p38 and p44/42 MAPKs were examined following 30 min-16 h of exposure. On the basis of these experiments, we chose to examine the 4-h time point for the experiments with the ET receptor antagonists and inhibitors of MAPK. Controls included untreated cells and/or cells treated with only ET-1 receptor antagonists or MAPK inhibitors alone. At the end of the experiment, the cells were lysed in 200 µl of 1% Triton X-100, and the protein content was determined using a bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Proteins (20 µg) were separated by SDS-PAGE on 10% polyacrylamide gels and then transferred to a nitrocellulose (NC) membrane (Bio-Rad Laboratories, Hercules, CA). Membranes were treated with either polyclonal COX-1 or COX-2 (1:1,000; Oxford Biochemical Research, Oxford, MI) or monoclonal antibodies to phosphorylated pp38 or ppERK (42/44) (Santa Cruz Biotechnology, Santa Cruz, CA), visualized with horseradish peroxidase-conjugated goat anti-IgG (1:2,000; Dako, Carpinteria, CA), and developed using Western blot chemiluminescence reagent (NEN Life Science Products, Boston, MA). After demonstration of pp38 or pERK, the NC membranes were stripped, and total MAPK levels were detected in the same membranes using p38 and ERK antibodies (Santa Cruz Biotechnology), respectively. Densitometric analysis of the resultant bands was carried out with a densitometer (Bio-Rad model GS-700) linked to a computer analysis system (Molecular Analysis Software; Bio-Rad).
RT-PCR analysis. For RT-PCR analysis of COX-1, COX-2, and ppET-1 gene expression, cells were harvested at baseline and 4 h after addition of 10 nM exogenous ET-1. Total cellular RNA was isolated with RNA STAT-60 (Teltest B, Friendswood, TX) following the manufacturer's instruction. RNA (4 µg) was subjected to reverse transcription as previously described (39). The sense and antisense primers for ppET-1 were 5'-TTGTGGCTTTCCAAGGAGCTCCAG-3' and 5'-GGTTGTCCCAGGCTTTCATG-3' (396 bp). The sense and antisense primers for COX-2 were 5'-TCCAGATCACATTTGATTGACA-3' and 5'-CTTTGACTGTGGGAG GATACA-3' (449 bp). The sense and antisense primers for COX-1 were 5'-TTC CAACCTTATCCCCAGCC-3' and 5'-CATGGCGATGCGGTTGC-3' (777 bp). The conditions for the PCR were as follows: after initial denaturation at 94°C for 2 min, the thermocycler (Hybaid OMN-E, Middlesex, UK) was programmed for 33 cycles: 1 min at 94°C, 1 min at 60°C (for ET-1)/55°C (for COX-2), and 2 min at 72°C. The reaction was concluded with a final extension step at 72°C for 8 min. PCR products were separated and visualized with 1.8% agarose gel containing 0.01% ethidium bromide. Optical density of the cDNA bands was determined by the computerized image-analysis system described above and normalized to RT-PCR products of human GAPDH (Clontech Laboratories, Palo Alto, CA) generated from each sample (7, 39).
Prostanoid measurements.
PLVSMC were treated with 10 nM ET-1 for 24 h. Measurements of the
stable metabolite of prostacyclin, 6-keto PGF1, and PGE2 were made by gas chromatography-mass spectrometry as
previously described (7). Quantification occurred by
stable isotope dilution, comparing the relative areas of the peaks of
endogenously produced eicosanoid and deuterated internal standard. The
results were normalized to ng/ml per 106 cells.
Measurement of intracellular ET-1. Intracellular levels of mature ET-1 were determined in homogenates of PLVSMC as previously described (3, 39). Briefly, the cells were washed in serum-free medium, trypsinized, and centrifuged at 1,000 rpm for 10 min. After homogenization of the cell pellet in 400 µl of 0.1% Triton-X in PBS, the homogenates were centrifuged and the supernatant was collected for solid-phase extraction by Sep-Pak C18 cartridges. Samples were dried under vacuum, and intracellular levels of mature ET-1 were determined by an ET-1 ELISA System (Biotrak ELISA System; Amersham International). The assay is sensitive to values of >1 fmol and cross-reacts with 100% of ET-1 and ET-2 (ET-2 is not generally found in the walls of the pulmonary vasculature), but not with ET-3 (<0.01% cross-reactivity). Each sample was run in duplicate. Results were expressed as femtomoles of ET-1 per 106 cells.
Statistics. Results are presented as means ± SE. Values from groups with different treatments were compared by either ANOVA followed by the Duncan multiple-comparison test or a paired Student's t-test. P values of <0.05 were considered significant. Analysis was performed using the Number Cruncher Statistical System (version 6.0.8).
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RESULTS |
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Characterization of PLVSMC.
Phase-contrast microscopy of subconfluent PLVSMC revealed that control
cells were stellate in shape (Fig.
1A). At confluence, the cells
were elongated in shape and formed "whorls" rather than the typical
"hill and valley" formation of cultured smooth muscle cells (Fig.
1B). Immunocytochemical techniques demonstrated that the
cells contained both smooth muscle -actin and smooth muscle myosin
and that these filaments generally colocalized. The density of both
filaments was most pronounced at the tips of the cytoplasmic processes
(Fig. 1, C and D).
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Effect of ET-1 on ppET-1 gene expression.
Time course experiments revealed that ET-1 caused a transient increase
in ppET-1 gene expression (n = 4). By RT-PCR, basal ppET-1 gene expression was low and exposure to ET-1 caused a gradual increase, peaking at 2 h and then falling to baseline by
16 h (Fig. 2). Pretreatment with
either the ETA (BQ-610) or the ETB (BQ-788)
receptor antagonists almost completely inhibited the ET-1-stimulated
increase in ppET-1 gene expression (Fig.
3): simultaneous pretreatment with both
receptor antagonists resulted in complete suppression.
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Effect of ET-1 on intracellular ET-1. ET-1 also caused an increase in newly synthesized intracellular ET-1 (n = 4). Basal intracellular levels of ET-1 were 11.8 ± 2.1 fmol/106 cells and, following 4-h incubation with ET-1, showed a fivefold increase (57.9 ± 2.6 fmol/106 cells, P < 0.05). To check that the increase in intracellular ET-1 levels was not due entirely to internalization, we treated sister cells with exogenous ET-1 for 4 h with and without pretreatment with the protein synthesis inhibitor BFA (50 µg/ml) for 15 min (2, 37). In the presence of BFA, basal levels of intracellular ET-1 were similar to untreated cells (13.4 ± 4.5 fmol/106 cells) and showed only a three- to fourfold increase in the presence of ET-1 (46.9 ± 1.5 fmol/106 cells), a value that was significantly less than that found in the ET-1-stimulated cells without pretreatment with BFA (P < 0.05). Thus, although PLVSMC internalize ET-1, synthesis of new protein occurs in response to ET-1 and accounts for nearly 20% of the increase.
ET-1 and COX-1 and COX-2 gene and protein expression.
PLVSMC express both the COX-1 and COX-2 genes at baseline, and their
expression is increased by exposure to ET-1 (n = 3). Time course experiments using RT-PCR demonstrated that expression of
the COX-1 and COX-2 genes peaked between 1 and 4 h and then fell
to basal values by 16 h (Fig.
4A). Western analysis also revealed a transient increase in expression of COX-1 and COX-2. ET-1
caused a gradual increase in COX-2 protein that peaked at 8 h and
then declined (Fig. 4B). ET-1-stimulated COX-1 expression was less striking than COX-2, showing peak expression at 2 h (Fig. 4).
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ET-1 and prostanoid release.
At baseline, levels of released PGE2 (54.2 ± 10.5 ng · ml1 · 106
cells
1) were higher than for 6-keto-PGF1
(13.3 ± 4.4 ng · ml
1 · 106
cells
1). Twenty-four hours of stimulation with ET-1
caused a significant increase in both PGE2 release
(91.5 ± 13.3 ng · ml
1 · 106
cells
1) and 6-keto-PGF1
(18.1 ± 4.2 ng · ml
1 · 106
cells
1, n = 5, P < 0.05).
ET-1 and the p38 and p42/44 MAPKs.
At baseline, Western analyses failed to detect the phosphorylated form
of pp38 MAPK and showed only low levels of pp44/42 MAPK. ET-1 treatment
resulted in an increase in phosphorylation of both the p38 and p42/44
MAPKs. Time course experiments revealed that pp38 expression peaked
between 4 and 8 h of exposure and pp44/42 expression peaked at
4 h (Fig. 5). Levels of
nonphosphorylated p38 and p44/42 MAPKs were unaffected by ET-1 (Fig.
5).
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MAPKs and ET-1-stimulated COX expression.
Pretreatment with either the inhibitor of p38 (SB-203580) or p44/42
(PD-98059) significantly inhibited ET-1-induced production of COX-2 (by
50 and 75%, respectively; P < 0.05, n = 4). Simultaneous pretreatment with both MAPK inhibitors downregulated
the ET-1-stimulated COX-2 induction, resulting in an additive effect
with 90% inhibition of the peak stimulated value. In contrast,
ET-1-stimulated COX-1 expression was not significantly altered by
either MAPK inhibitor (Fig. 6, A and
B).
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ET-1 receptors and ET-1-stimulated COX expression.
Pretreatment with the ETA antagonist BQ-610 caused a 50%
decrease in ET-1-stimulated COX-2 expression, whereas the
ETB antagonist BQ-788 had little effect. Simultaneous
treatment with both receptor antagonists had a similar effect to that
for BQ-610 alone (Fig. 7A, n = 4). The effect of both inhibitors on
ET-1-stimulated COX-1 expression was similar, but the effect of BQ-610
was less impressive (Fig. 7B).
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DISCUSSION |
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The present study demonstrates that PLVSMC isolated from control
sheep can be maintained in culture and that although they exhibit both
smooth muscle -actin and smooth muscle myosin, they exhibit a
different morphology compared with the spindle-shaped main pulmonary
artery smooth muscle cells with a typical hill-and-valley appearance.
Our study also shows that PLVSMC express COX-1 and COX-2 and ppET-1
genes and proteins at baseline. Furthermore, we demonstrate that
exogenous ET-1 results in a transient increase in ppET-1 gene
expression and that activation of both the ETA and
ETB receptors regulate this increase. We also show a
striking fivefold increase in levels of intracellular ET-1, and
experiments with BFA indicate that ~25% of the increase is
attributable to new synthesis, the remaining 75% being the result of
internalization of extracellular ET-1. Stimulation with ET-1 also
results in transient increases in the COX-1 and COX-2 genes and
proteins that are associated with increased release of the vasodilator
prostaglandins PGE2 and prostacyclin. In addition, we found
that ET-1-stimulated COX-2, but not COX-1, expression is regulated by
the ETA receptor; the ETB receptor had little
effect on either COX. ET-1 also causes phosphorylation of the p38 and
p44/42 MAPKs, and their inhibition results in suppression of
ET-1-stimulated COX-2, but not COX-1, expression.
A delicate balance between vasoconstrictor and vasodilator agents
exists in the small pulmonary arteries responsible for normal homeostasis. It is well established that the endothelial cell is a
source of many of these vasoactive agents (e.g., prostaglandins, ET-1,
and nitric oxide), but few studies have addressed the contribution of
other cells in the walls of these small arteries. The present study
demonstrates that both vasoconstrictor and vasodilator agents are
synthesized by PLVSMC. In previous studies, we demonstrated that ET-1
is synthesized in smooth muscle cells isolated from control sheep main
and midregion pulmonary artery and suggested that these cells may
contribute to local arterial tone (3, 4). We also
demonstrated that ppET-1 gene expression was regulated by exogenous
ET-1, as has also been shown in rat endothelial and vascular smooth
muscle cells (18, 25). The present study extends these
findings to the precursor smooth muscle cells of the peripheral pulmonary arteries and shows that ET-1 stimulates ppET-1 gene expression, and, in addition, our data document intracellular synthesis
of a new peptide in these cells. Thus it seems that ET-1 is synthesized
in smooth muscle-type cells along the entire pulmonary arterial tree
and that local production of ET-1 by PLVSMC, as well as endothelial
cells, may contribute to the local maintenance of normal vascular tone
in the peripheral pulmonary arteries. Our finding of both smooth muscle
-actin and smooth muscle myosin is also in line with this notion.
As mentioned above, the endothelial cell has been considered the major player in synthesis and release of vasoactive prostaglandins; for example, under physiological conditions, constitutive COX-1 is highly localized to the endothelium (7). However, under pathological conditions, expression of COX-2 is induced in many cells, including pulmonary artery smooth muscle (36), whereas only minor changes occur in expression of COX-1 (33). The present study demonstrates that PLVSMC from control sheep exhibit basal COX-1 and COX-2 gene and protein expression and synthesize PGE2 and prostacyclin and, furthermore, that ET-1 upregulates expression of both enzymes and increases release of both PGE2 and prostacyclin. To our knowledge, this is the first report demonstrating that ET-1 regulates both COX-1 and COX-2. Thus it seems that PLVSMC contribute to local basal production of both vasodilator prostaglandins and ET-1. In this model, paracrine release of ET-1 first increases and then induces COX-derived release of prostaglandins in a negative feedback loop.
ET-1 binds with high affinity to its two G-coupled receptors, ETA and ETB, to elicit a wide range of intracellular pathways and cellular functions (11, 24, 36). ETA receptors are expressed abundantly on vascular smooth muscle cells (24). Two types of ETB receptors have been identified: ETB2 receptors are expressed on vascular smooth muscle cells and mediate vasoconstriction; ETB1 receptors are expressed on endothelial cells and contribute to vasodilatation at least in part through release of nitric oxide and prostacyclin (9, 10, 33). In the human pulmonary artery, the ETA receptors have been found to predominate in arteries from 0.5 to >8 mm in diameter, although the number of ETB receptors increases with decreasing arterial diameter (3). These data correlate well with contraction assays showing that ET-1-induced contraction of rat proximal arteries is dependent on ETA receptors, whereas in human and rat distal arteries of 150-250 µm in diameter, ETB is responsible (27, 28). The present study indicates that both receptors are present on normal sheep PLVSMC and that both receptors regulate expression of ppET-1. This contrasts with an earlier study using rat glomerular cells, in which only ETB was found to be responsible for regulation of ppET-1 mRNA (25). Whether this difference is due to cell and tissue type is not certain. Together these data suggest that the ETB receptors of the endothelial cells initiate contraction in small arteries of the lung but that activation of both the ETA and ETB receptors on the subendothelial PLVSMC contributes to the sustained constriction.
Activation of the ETB receptor has been linked to activation of phospholipase A2, release of arachidonic acid, and production of both vasodilator and constrictor prostaglandins (26, 42). Our data demonstrate that the ETA receptor mainly contributes to regulation of ET-1-stimulated COX-2 expression in PLVSM. The reason for this difference is obscure but may involve use of different organs and species. Whether regulation of COX-1 by ETB is a direct effect is not certain, but our data using inhibitors suggest that the downstream MAPKs are involved.
The intracellular signaling cascades underlying activation of the ET-1 receptors involve G protein-mediated activation of various kinases and phosphorylation of MAPKs (36). The latter participate in cellular events associated with cell growth and differentiation via transcriptional activation (1, 23, 33). ET-1 is known to activate the MAPK subfamilies (including p42/44 and p38) (34), and our data in PLVSMC confirm this finding. Our data also add to previous findings indicating a role for the MAPKs in the induction of COX-2 in endothelial cells (35), keratinocytes (8), and renal cells (16) stimulated by agents other than ET-1. Furthermore, our data show that this phosphorylation is linked to ET-1-stimulated expression of COX-2. Thus it would seem that, following activation of the ETA receptor, upregulation of COX-2 expression involves phosphorylation of the p38 and p42/44 MAPKs. Regulation of ET-1-stimulated COX-1 expression, on the other hand, is independent of the ET receptors and MAPKs, and its expression is likely to be stimulated by an event downstream of receptor activation.
In summary, our data demonstrate that PLVSMC exhibit basal expression of ppET-1, COX-1, and COX-2 and release the vasodilator prostaglandins, prostacyclin, and PGE2. Furthermore, exogenous ET-1 upregulates expression of each of these genes and proteins and increases release of prostaglandins. Our data also show that ET-1-stimulated upregulation of ppET-1 involves activation of both the ETA and ETB receptors, whereas upregulation of COX-2 is mediated via activation of ETA alone; ET-1-stimulated increase in COX-1 expression was ET receptor independent. In addition, we showed that ET-1 caused phosphorylation of p38 and p44/42, and inhibition of these MAPKs suppressed the ET-1-stimulated increase in COX-2, but not COX-1. We conclude that vasoactive mediators derived from both PLVSMC and endothelial cells regulate the normal basal tone of the intra-acinar pulmonary arteries.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-48536.
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FOOTNOTES |
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Address for reprint requests and other correspondence: B. Meyrick, Center for Lung Research, MCN T-1217, Nashville, TN 37232-2650 (E-mail: barbara.meyrick{at}vanderbilt.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 10, 2003;10.1152/ajplung.00215.2002
Received 3 July 2002; accepted in final form 15 November 2002.
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