Departments of 1 Pediatrics and 3 Molecular Pharmacology, Northwestern University Medical School, Chicago, Illinois 60611-3008; and 2 Department of Pediatrics, University of California, San Francisco, California 94143-0106
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have shown that increased pulmonary blood flow at birth increases the activity and expression of endothelial nitric oxide (NO) synthase (eNOS). However, the signal transduction pathway regulating this process is unclear. Because protein kinase C (PKC) has been shown to be activated in response to shear stress, we undertook a study to examine its role in mediating shear stress effects on eNOS. Initial experiments demonstrated that PKC activity increased in response to shear stress. NO production in response to shear stress was found to be biphasic, with an increase in NO release up to 1 h, a plateau phase until 4 h, and another increase between 4 and 8 h. PKC inhibition reduced the initial rise in NO release by 50% and the second increase by 70%. eNOS mRNA and protein levels were also increased in response to shear stress, whereas PKC inhibition prevented this increase. The stimulation of PKC activity with phorbol ester increased eNOS gene expression without increasing NO release. These results suggest that PKC may play different roles in shear stress-mediated release of NO and increased eNOS gene expression.
signal transduction; gene regulation; pulmonary blood flow; nitric oxide synthase; protein kinase C
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
AFTER BIRTH, WITH INITIATION of ventilation by the lungs, pulmonary vascular resistance decreases and pulmonary blood flow increases 8- to 10-fold to match systemic blood flow (7, 11, 21, 45). This process is regulated by a complex and incompletely understood interplay between mechanical and metabolic factors (15). Both rhythmic distension of the fetal lamb lung and ventilation without oxygenation produce partial pulmonary vasodilation (11). There is also release of vasoactive substances that decrease pulmonary vascular resistance and increase pulmonary blood flow (29).
There is increasing evidence that the vasodilator nitric oxide (NO)
mediates pulmonary vascular tone in the perinatal period (1, 9,
16, 33, 50). First, in fetal lambs, acetylcholine decreases
pulmonary vascular resistance and increases pulmonary blood flow
(49); these hemodynamic effects can be blocked by N-nitro-L-arginine
(L-NNA) or other L-arginine analogs that
inhibit NO synthesis (49). Second, inhibition of NO
synthesis increases pulmonary vascular resistance in fetal lambs and
attenuates the increase in pulmonary blood flow induced by maternal
hyperbaric oxygen exposure (1, 9, 33, 50). Third,
L-arginine, the precursor of NO, increases pulmonary blood
flow in fetal and newborn lambs and augments endothelium-dependent
pulmonary vasodilation (1, 13). Fourth, inhaled NO
decreases pulmonary vascular resistance in fetal lambs and in newborn
lambs with pulmonary hypertension (23, 54). Fifth,
methylene blue, an inhibitor of soluble guanylate cyclase, increases
pulmonary vascular resistance, whereas M & B 22948 and dipyridamole,
inhibitors of cGMP phosphodiesterase, decrease pulmonary vascular
resistance and augment endothelium-dependent pulmonary vasodilation in
fetal and newborn lambs (6, 14). Several studies have also
investigated the potential role of NO in the developing pulmonary
circulation. Both basal and agonist-stimulated endothelial production
of NO have been demonstrated in the fetal, newborn, and adult pulmonary
vasculature (47). Basal NO production rises twofold from
late gestation to 1 wk of life and another 1.6-fold from 1 to 4 wk of
life in intrapulmonary arteries (47). A physiological
study (47) of intrapulmonary arteries and isolated lung
preparations of the sheep revealed maturational increases in
NO-mediated relaxation during the late fetal and early postnatal periods. Similarly, in the pig, there is an increase in NO-mediated relaxation during the first 2 wk of life, followed by a decrease (41, 55). Coinciding with this physiological data,
endothelial NO synthase (eNOS) mRNA and protein increase in late
gestation and decrease postnatally in rat and sheep lung parenchyma
(17, 22, 40). In addition, lung mRNA levels for the
1- and
1-subunits of soluble guanylate
cyclase during late-gestation fetal and neonatal rats are sevenfold
higher than those found in the adult rat (5). This
maturational increase in NO and cGMP production parallels the dramatic
decrease in pulmonary vascular resistance that occurs at birth. Also,
in the late-gestation fetal lamb, an infusion of the eNOS inhibitor
L-NNA markedly attenuates the increase in pulmonary blood
flow associated with ventilation at birth (1, 16). Taken
together, these data strongly suggest that NO activity mediates, in
part, the fall in pulmonary vascular resistance during the transitional
pulmonary circulation and maintains the normal low postnatal pulmonary
vascular resistance.
At least one important mechanism by which pulmonary vasodilation occurs relates to the increase in shear stress on the pulmonary vascular endothelium that is induced by the increase in pulmonary blood flow. Fluid shear stress is defined as the tractive force produced by moving a viscous fluid (blood) on a solid body (vessel wall), constraining its motion. The magnitude of shear stress increases with fluid viscosity (37). When endothelial cells (ECs) are subjected to shear stress, diverse responses are initiated, some of which occur within minutes and others that develop over many hours or days. Rapid changes occur in ionic conductance, adenylate cyclase activity, inositol trisphosphate generation, and intracellular free calcium levels. If the shear stress is maintained, the rapid responses are then followed by much slower changes in gene expression and by structural reorganization of the cytoskeleton, producing changes in cell shape (36). NOS activity, NO production, and eNOS mRNA and protein expression are increased when ECs are exposed to increased shear stress (24, 27, 28, 38, 39, 42, 52, 53).
Previously, Black et al. (3) and Fineman et al. (16) have shown that the increased pulmonary blood flow occurring at birth leads to an increase in eNOS expression in the pulmonary vascular endothelium and that the NO released mediates, in part, both the immediate and the more gradual decreases in pulmonary vascular resistance after birth. However, the components of the second messenger system that transduce this signal are incompletely understood. Thus the purpose of this study was to begin to elucidate this signal transduction pathway to identify the shear stress-induced molecular events that regulate eNOS gene expression. We chose to investigate the role of the protein kinase C (PKC) system in transducing the shear stress signal because increasing evidence has shown that this pathway can be stimulated in ECs exposed to shear stress (26, 28, 39, 42, 52). Here, we demonstrate PKC-mediated changes in eNOS gene expression in response to laminar shear stress.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture techniques. The heart and lungs were obtained from fetal (138-140 days gestation) lambs after death. These fetal lambs had not undergone previous surgery or study. The main and branching pulmonary arteries were removed and dissected free, and the adventitia was removed. The exterior of the vessel was rinsed with 70% ethanol. The vessel was then opened longitudinally, and the interior was rinsed with PBS to remove any blood. With a cell scraper, the endothelium was lightly scraped away, placed in medium DME-H16 (with 10% fetal bovine serum and antibiotics), and incubated at 37°C in 21% O2-5% CO2-balance N2. After 5 days, islands of ECs were cloned to ensure purity. Basic fibroblast growth factor (1 ng/ml; a gift from Dr. Denis Gospodarowicz, Chiron, Emeryville, CA) was added to the medium every other day. When confluent, the cells were passaged to maintain them in culture or frozen in liquid nitrogen. EC identity was confirmed by the typical cobblestone appearance, contact inhibition, specific uptake of acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-AcLDL, Molecular Probes, Eugene, OR), and positive staining for von Willebrand factor (DAKO, Carpinteria, CA). Ovine fetal pulmonary arterial ECs (OFPAECs) were studied between passages 3 and 10.
Fluid shear stress.
A cone-plate viscometer similar to that described by Sdogous et al.
(46) was designed and built such that it accepts 15-cm tissue culture plates. This apparatus consisted of a cone of shallow angle () rotating at angular velocity (
) on top of a tissue culture plate containing medium of a known viscosity (µ) on which ECs
were present as a monolayer. This allowed the monolayer to be subjected
to a radially constant fluid shear stress (
;
dyn/cm2) as calculated with the formula
= µ
/
. A cone angle of 0.5° was used to achieve laminar flow
rates representing levels of shear stress within physiological
parameters. Typical physiological shear stress in the major human
arteries is in the range of 5-20 dyn/cm2
(31), with localized increases to 30-100
dyn/cm2 (12). Thus we imparted a shear stress
of 20 dyn/cm2 to mimic the upper limit of the physiological
range. Also, because a previous study (39) showed that
pulsatile and laminar flow appear to induce similar effects on NO
production, laminar shear stress was employed.
Generation of ovine eNOS cDNA and antiserum. Oligonucleotides were synthesized (with bovine eNOS as a template) to allow amplification of this region within the ovine eNOS sequence. The sequences of the oligonucleotides were 5'-CCTCCGGAGGGGCCCAAGTTCCCTCGC-3' for oligonucleotide 1 and 5'-CACGTCGAAGCGCCGTTTCCGGGGGT-3' for oligonucleotide 2. The region amplified corresponds to amino acids 62-288 of the heme-binding domain of the eNOS protein (48). Total RNA prepared from ovine fetal lung was used in RT-PCRs (kit from PerkinElmer, Foster City, CA). The cDNA fragment generated (681 bp) was then cloned directly into the pCR II vector (Invitrogen, San Diego, CA), sequenced (Sequenase kit from USB, Cleveland, OH), and then subcloned into pBluescript KS+ (Stratagene, La Jolla, CA). The sequence in this region for ovine eNOS is 96.6% identical to bovine eNOS.
RNA isolation and analysis.
OFPAECs were grown on 15-cm plates and lysed with 4 M guanidinium
thiocyanate. Total RNA was isolated by acid-phenol extraction (8). Because of its relatively low abundance, eNOS
expression was evaluated by RNase protection assay according to our
standard procedures (3). Briefly, single-strand antisense
cRNA probes were synthesized, and then 50 µg of total RNA were
hybridized overnight to 500,000 counts/min (cpm) of probe in 80%
formamide-40 mM PIPES-0.4 M NaCl-1 mM EDTA at 42°C. This was followed
by digestion with 5-10 U of RNase A and 25 U of RNase T1,
phenol-CHCl3 extraction, and ethanol precipitation.
Protected fragments were then separated by electrophoresis on a
DNA-sequencing gel and exposed to film at 70°C. Also included was a
170-bp cDNA fragment of ovine 18S to control for RNA input and recovery
(3, 4).
Protein preparation for Western blotting. Cells were harvested in ice-cold PBS, centrifuged, resuspended in PBS, and sonicated before protein quantitation. One hundred micrograms of each protein extract were run on reducing SDS-polyacrylamide gels. The gels were electrophoretically transferred to Hybond-polyvinylidene difluoride membranes, blocked in PBS-Tween (0.1%) containing 5% nonfat dry milk, and incubated with an eNOS monoclonal antibody (1:2,500 dilution; Transduction Laboratories). After being washed to remove excess primary antibody, the membranes were incubated with a secondary goat anti-mouse IgG conjugated with horseradish peroxidase. After being washed to remove excess secondary antibody, the bands were visualized with chemiluminescence procedures (3, 4).
Measurement of released nitrate. In solution, NO reacts with molecular oxygen to form nitrite and with oxyhemoglobin and superoxide anion to form nitrate (NOx). The nitrite and NOx were reduced with vanadium(III) and hydrochloric acid at 90°C. NO was then purged from the solution, resulting in a peak of NO. Therefore, this value represents total NO, nitrite, and NOx. This peak was then detected by chemiluminescence (NOA 280, Sievers Instruments, Boulder, CO). The detection limit was 1 nM/ml of NOx.
Inhibition and stimulation and analysis of PKC activity. PKC activity was determined with a commercially available kit (Amersham).
Statistical analysis. Quantitation of autoradiographic results was performed by scanning (Microtek E6, Microtek, Compton, CA) the bands of interest into an image-editing software program (Adobe Photoshop, Adobe Systems, Mt. View, CA). Band intensities from the RNase protection assays and the Western blot analysis were analyzed densitometrically on a Macintosh computer (model 9500, Apple Computer, Cupertino, CA) with the public domain NIH Image program (developed at the National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image). In the RNase protection assays, to control for the amount of input RNA and the recovery of protected fragments, the mRNA signal of interest was normalized to the corresponding 18S signal for each lane. Results from unsheared cells were assigned the value of 1 (relative mRNA of interest). For Western blot analysis, to ensure equal protein loading, duplicate polyacrylamide gels were run. One was stained with Coomassie blue. Results from unsheared cells were assigned the value of 1 (relative protein of interest). The means ± SD were calculated for the relative mRNAs and proteins for each condition and were compared with the unpaired t-test, with the use of the GB-STAT software program. P < 0.05 was considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
After isolating the relevant late-gestation
OFPAECs, we exposed them to a controlled level of shear
stress with a cone-plate viscometer similar to that described by
Sdogous et al. (46). Initial experiments with 20 dyn/cm2 on OFPAECs demonstrated that the cells changed
morphology from a cobblestone appearance to a more elongated shape, a
feature of ECs under shearing conditions (Fig.
1). We next measured cellular PKC
activity in OFPAECs exposed to shear stress (20 dyn/cm2 for
8 h). PKC activity was significantly increased (7.9-fold, P < 0.05; Fig. 2).
However, PKC activity was not increased when cells were exposed
to shear stress (20 dyn/cm2 for 8 h) in the presence
of the PKC inhibitor staurosporine (1 µM).
|
|
Next, we isolated RNA and protein from OFPAECs exposed to 20 dyn/cm2 for 0-8 h in the presence and absence of PKC
inhibition to determine the effect on eNOS gene expression. The
expression of eNOS mRNA was then determined with the RNase protection
assay (Fig. 3, A and
B) and eNOS protein was determined by Western blot
analysis (Fig. 3, C and D). The expression of
eNOS was found to increase in response to increased shear stress. Both
eNOS mRNA (4.9-fold at 8 h; Fig. 3, A and B)
and eNOS protein (2.9-fold at 8 h; Fig. 3, C and
D) were induced by fluid shear stress. However, when PKC
activity was attenuated with the specific PKC inhibitor calphostin C
(50 nM), the shear stress-induced increase in eNOS gene expression was
attenuated (Fig. 3). When other PKC inhibitors were used
(staurosporine, H-7, and bisindolylmaleimide), similar results were
obtained (data not shown), implicating PKC in the transduction of the
shear stress signal transduction pathway, leading to an increase in
eNOS gene expression.
|
Because NOS activity and hence NO production are increased when ECs are
exposed to increased shear stress, we then determined if PKC inhibition
would affect NO release from sheared OFPAECs. The results
obtained demonstrated that there was a biphasic rise in NO production
in OFPAECs exposed to shear stress, with an initial rapid increase in
NO release (0-1 h), a plateau phase (1-4 h), and then another
increase in NO release (4-8 h; Fig.
4). This second increase in NO release
corresponded to the time at which we could measure a significant
increase in new eNOS protein (Fig. 3, C and D).
Furthermore, NO release was reduced to a greater extent at 4-8 h
compared with 0-1 h after PKC inhibition (Fig. 4B).
|
Finally, we determined whether the stimulation of cellular PKC activity
altered either eNOS gene expression or eNOS activity in the absence of
shear stress. When OFPAECs were exposed to the PKC activator phorbol
12-myristate 13-acetate (PMA; 300 nM) for 4 h, the expression of
eNOS mRNA was shown to increase by 3.1-fold over that in
cells treated with vehicle alone (P < 0.05; Fig. 5). However,
there was no concomitant increase in the level of NO released from the
PMA-treated cells compared with those exposed to vehicle alone (Fig.
6A). This lack of NO release
from PMA alone did not appear to be a result of a PKC-mediated
inhibition of the eNOS enzyme because OFPAECs pretreated with PMA
before exposure to shear stress produced equivalent levels of NO to
those exposed to shear alone (Fig. 6B). These results
suggest that the role of PKC may be different in the signal
transduction pathways that are activated by shear stress to stimulate
NO production or eNOS gene expression.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the fetus, pulmonary vascular resistance is high, and pulmonary blood flow is low. At birth, there is a rapid 8- to 10-fold increase in pulmonary blood flow related to a marked decrease in pulmonary vascular resistance. These rapid changes in pulmonary blood flow and resistance are then followed by more gradual changes that occur during the next several hours. There is increasing evidence that the changes in pulmonary vascular tone are mediated by NO, possibly in response to increased shear stress on the pulmonary vascular endothelium (17, 21, 40, 41, 47). In a number of clinical conditions, there is a failure of the pulmonary circulation to undergo the normal transition to postnatal life, resulting in persistent pulmonary hypertension of the newborn (2, 4, 35). A better understanding of the mechanisms responsible for the pulmonary vascular changes at birth may lead to new prevention of and improved treatment strategies for persistent pulmonary hypertension of the newborn. However, the signaling mechanisms involved in mediating these changes remain unclear.
To allow a molecular analysis of the effects of birth on the fetal pulmonary endothelium, we first isolated and cultured the relevant cells exposed to increased shear stress at birth. These cells were late-gestation OFPAECs (135-140 days gestation). To mimic the effects of increased blood flow, we used a cone-plate viscometer that allowed us to expose the OFPAEC monolayer to a controlled level of shear stress. The laminar shear stress produced by this technique has previously been shown (39) to induce NO production and to upregulate the level of eNOS mRNA in human umbilical vein ECs (HUVECs). Our results indicate a biphasic production of NO by OFPAECs in response to shear stress. The initial increase in NO production between 0 and 1 h may have been due to the activation of preformed eNOS enzyme. In contrast, the second phase of NO production, occurring after 6-8 h of shear stress, corresponded with an increase in levels of eNOS mRNA and protein and may have been stimulated by factors that regulate eNOS gene expression. The eNOS gene 5'-flanking region contains a cis-acting regulatory sequence identical to the previously identified shear stress-responsive element (43), raising the possibility that shear stress-induced factors may control eNOS gene transcription via this potential shear stress-responsive element.
Shear stress has been shown to regulate the expression of a variety of
genes that encode proteins affecting transcription, cytoskeletal
structure, apoptosis, release of vasoactive substances, smooth
muscle proliferation, and EC growth arrest (10, 31). Recently, the effects of shear stress on the endothelin system were
studied in HUVECs (34). This work demonstrated a
downregulation of endothelin gene expression by shear stress, which was
prevented by the eNOS inhibitor
N-nitro-L-arginine methyl
ester. It seems likely that the NO and endothelin systems
regulate each other via an autocrine feedback loop (32).
These data identify a role for shear stress in the control of gene
expression within the NO and endothelin cascades and further illustrate
the complex interactions between them. However, the precise mechanisms
involved remain to be determined.
Increasing evidence has shown that the PKC system is stimulated in ECs exposed to shear stress (26, 28, 39, 42, 52). We thus undertook a study to determine if shear stress-stimulated PKC activity can transduce the signal to activate eNOS. The results obtained clearly demonstrated that the PKC system was both necessary and sufficient to induce eNOS gene expression. However, although PKC was found to play an important part in the stimulation of NO release, both initially and after 6-8 h of exposure to shear stress, PMA-induced activation of PKC alone was insufficient to activate the eNOS enzyme. PKC activation is thought to occur in response to the synergistic action of diacylglycerol and calcium, both of which can be generated by signal-induced hydrolysis of membrane phospholipids. Thus intracellular calcium might be increased on exposure of OFPAECs to PMA. Because eNOS is calcium dependent, it might have been expected that NO production would be stimulated. However, we did not obtain this result. Because a previous study (18) suggested that PKC-mediated phosphorylation may inhibit eNOS activity, it was possible that PMA was inhibiting eNOS, reducing its ability to produce NO. However, this was not the case because OFPAECs produced similar amounts of NO whether cells were sheared in the presence or absence of PMA. Our results suggest that PKC may have two distinct roles in the transduction of the shear stress signal, leading to NO production in OFPAECs. The contribution by PKC to the initial response that results in increased eNOS enzyme activity may be different from its contribution in the longer-term response that results in increased eNOS gene expression.
PKC can be measured in both cytosolic and particulate fractions, and
different tissues appear to have characteristic patterns of expression
of PKC isozymes, leading to the hypothesis that each isozyme has a
different biological role. Our results have identified PKC as one
signal transduction molecule involved in regulating eNOS activity and
gene expression by fluid shear stress. In fact, shear stress has been
shown to elevate PKC- immunostaining in HUVECs, with increased
translocation to the cell membrane (19). Furthermore, it
has been demonstrated that PKC-
is activated by shear stress and
plays a role in transducing the shear stress signal, resulting in the
activation of extracellular signal-regulated kinase 1/2
(51), whereas PKC-dependent activation of extracellular signal-regulated kinase 1/2 has also been shown in bovine aortic ECs in
response to cyclic strain (20). A previous study
(30) found that PMA incubation produced the translocation
of PKC-
and PKC-
from the cytosol to the cell membrane in HUVECs,
indicating activation of these isoforms. When these cells were
transfected with a 3.5-kb fragment of the human NOS III promoter
driving a luciferase reporter gene, PMA stimulated promoter activity up to 2.5-fold (30). In addition, PMA enhanced basal and
bradykinin-stimulated NO production and stimulated eNOS mRNA expression
in a concentration- and time-dependent manner (30).
Specific PKC inhibitors also prevented the upregulation of NOS III mRNA
produced by PMA. These data suggest that stimulation of PKC is involved
in the signaling pathway activating the human eNOS gene promoter in
human endothelium.
The expression of PKC isoforms has been studied in bovine aortic ECs
with immunohistochemical and Western blotting techniques (44,
51) and in cerebral ECs with RT-PCR. The results obtained are
interesting because each cell type appears to express a different spectrum of PKC isozymes: bovine aortic ECs have been shown to express
the PKC-, -
, -
, and -
isoforms, whereas cerebral ECs expressed the
,
,
,
, and
isoforms. These results
suggest that ECs can express a different complement of PKC isoforms in different areas of the circulation. The PKC expression pattern of the
ECs of the pulmonary vasculature has not been determined and may thus
be important in identifying the mechanisms that induce eNOS expression
in response to increased fluid shear stress.
In summary, the involvement of PKC in shear stress-induced NO production appears to occur at the level of eNOS enzyme activity and eNOS gene expression. It seems likely that a subset of PKC isoforms provides distinct functions, giving rise to several different signaling pathways. As yet, these isoforms and their specific roles remain to be elucidated. In addition, the PKC-independent pathways that also result in NO production in response to shear stress have yet to be determined. Further studies are required to identify the mechanisms involved.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Jeff Fineman (University of California, San Francisco, CA) for help with the statistical analyses.
![]() |
FOOTNOTES |
---|
This work was supported in part by National Institutes of Health Grants HL-60190, HD-39110, and HD-36809; March of Dimes Grant FY00-98; and Grant-in-Aid 0051409Z0 from the Midwest Affiliates of the American Heart Association (all to S. M. Black).
S. M. Black is a member of the Feinberg Cardiovascular Research Institute.
Address for reprint requests and other correspondence: S. M. Black, Div. of Neonatology, Northwestern Univ. Medical School, Ward 12-191, 303 E. Chicago Ave., Chicago, IL 60611-3008 (E-mail: steveblack{at}northwestern.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.
Received 25 October 2000; accepted in final form 22 March 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abman, SH,
Chatfield BA,
Hall SL,
and
McMurtry IF.
Role of endothelium-derived relaxing factor during transition of pulmonary circulation at birth.
Am J Physiol Heart Circ Physiol
259:
H1921-H1927,
1990
2.
Abman, SH,
Shanley PF,
and
Accurso FJ.
Failure of postnatal adaptation of the pulmonary circulation after chronic intrauterine pulmonary hypertension in fetal lambs.
J Clin Invest
83:
1849-1858,
1989[ISI][Medline].
3.
Black, SM,
Johengen MJ,
Ma ZD,
Bristow J,
and
Soifer SJ.
Ventilation and oxygenation induce endothelial nitric oxide synthase gene expression in the lungs of fetal lambs.
J Clin Invest
100:
1448-1458,
1997
4.
Black, SM,
Johengen M,
and
Soifer S.
Coordinated regulation of genes of the nitric oxide and endothelin pathways during the development of pulmonary hypertension in fetal lambs.
Pediatr Res
44:
821-830,
1998[Abstract].
5.
Bloch, KD,
Filippov G,
Sanchez LS,
and
Nakane M.
Pulmonary soluble guanylate cyclase, a nitric oxide receptor, is increased during the perinatal period.
Am J Physiol Lung Cell Mol Physiol
272:
L400-L406,
1997
6.
Braner, DA,
Fineman JR,
Chang R,
and
Soifer SJ.
M & B 22948, a cGMP phosphodiesterase inhibitor, is a pulmonary vasodilator in lambs.
Am J Physiol Heart Circ Physiol
264:
H252-H258,
1993
7.
Cassin, S,
Dawes GS,
Mott JC,
Ross BB,
and
Strang LB.
The vascular resistance of the foetal and newly ventilated lung of the lamb.
J Physiol (Lond)
171:
61-79,
1964[ISI].
8.
Chomczynski, P,
and
Sacchi N.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:
156-159,
1987[ISI][Medline].
9.
Cornfield, DN,
Chatfield BA,
McQueston JA,
McMurtry IF,
and
Abman SH.
Effects of birth-related stimuli on L-arginine-dependent pulmonary vasodilation in ovine fetus.
Am J Physiol Heart Circ Physiol
262:
H1474-H1481,
1992
10.
Davies, PF,
Barbee KA,
Volin MV,
Robotewskyj A,
Chen J,
Joseph L,
Griem ML,
Wernick MN,
Jacobs E,
Polacek DC,
dePaola N,
and
Barakat AI.
Spatial relationships in early signaling events of flow-mediated endothelial mechanotransduction.
Annu Rev Physiol
59:
527-549,
1997[ISI][Medline].
11.
Dawes, GS,
Mott JC,
Widdicombe JG,
and
Wyatt DG.
Changes in the lungs of the newborn lamb.
J Physiol (Lond)
121:
141-162,
1953[ISI].
12.
Dewey, CF,
Bussolari SR,
Gimbrone MA,
and
Davies PF.
The dynamic response of vascular endothelial cells to fluid shear stress.
J Biomech Eng
103:
177-185,
1981[ISI][Medline].
13.
Fineman, JR,
Chang R,
and
Soifer SJ.
L-Arginine, a precursor of EDRF in vitro, produces pulmonary vasodilation in lambs.
Am J Physiol Heart Circ Physiol
261:
H1563-H1569,
1991
14.
Fineman, JR,
Crowley MR,
Heymann MA,
and
Soifer SJ.
In vivo attenuation of endothelium-dependent pulmonary vasodilation by methylene blue in the newborn lamb.
J Appl Physiol
71:
735-741,
1991
15.
Fineman, JR,
Soifer SJ,
and
Heymann MA.
Regulation of pulmonary vascular tone in the perinatal period.
Annu Rev Physiol
57:
115-134,
1995[ISI][Medline].
16.
Fineman, JR,
Wong J,
Morin FC,
Wright L,
and
Soifer SJ.
Chronic nitric oxide inhibition in utero produces persistent pulmonary hypertension in newborn lambs.
J Clin Invest
93:
2675-2683,
1994[ISI][Medline].
17.
Halbower, AC,
Tuder RM,
Franklin WA,
Pollock JS,
Forstermann U,
and
Abman SH.
Maturation-related changes in endothelial nitric oxide synthase immunolocalization in developing ovine lung.
Am J Physiol Lung Cell Mol Physiol
267:
L585-L591,
1994
18.
Hirata, K,
Kuroda R,
Sakoda T,
Katayama M,
Inoue N,
Suematsu M,
and
Kawashima S.
Inhibition of endothelial nitric oxide synthase activity by protein kinase C.
Hypertension
25:
180-185,
1995
19.
Hu, YL,
and
Chien S.
Effects of shear stress on protein kinase C distribution in endothelial cells.
J Histochem Cytochem
45:
237-249,
1997
20.
Ikeda, M,
Takei T,
Mills I,
Kito H,
and
Sumpio BE.
Extracellular signal-regulated kinases 1 and 2 activation in endothelial cells exposed to cyclic strain.
Am J Physiol Heart Circ Physiol
276:
H614-H622,
1999
21.
Iwamoto, HS,
Teitel D,
and
Rudolph AM.
Effects of birth-related events on blood flow distribution.
Pediatr Res
22:
634-640,
1987[Abstract].
22.
Kawai, N,
Bloch DB,
Filippov G,
Rabkina D,
Suen HC,
Losty PD,
Janssens SP,
Zapol WM,
de la Monte S,
and
Bloch KD.
Constitutive endothelial nitric oxide synthase gene expression is regulated during lung development.
Am J Physiol Lung Cell Mol Physiol
268:
L589-L595,
1995
23.
Kinsella, JP,
McQueston JA,
Rosenberg AA,
and
Abman SH.
Hemodynamic effects of exogenous nitric oxide in ovine transitional pulmonary circulation.
Am J Physiol Heart Circ Physiol
263:
H875-H880,
1992
24.
Korenaga, R,
Ando J,
Tsuboi H,
Yang W,
Sakuma I,
Toyooka T,
and
Kamiya A.
Laminar flow stimulates ATP- and shear stress-dependent nitric oxide production in cultured bovine endothelial cells.
Biochem Biophys Res Commun
198:
213-219,
1994[ISI][Medline].
25.
Krizbai, I,
Szabo G,
Deli M,
Maderspach K,
Lehel C,
Olah Z,
Wolff JR,
and
Joo F.
Expression of protein kinase C family members in the cerebral endothelial cells.
J Neurochem
65:
459-462,
1995[ISI][Medline].
26.
Kuchan, MJ,
and
Frangos JA.
Shear stress regulates endothelin-1 release via protein kinase C and cGMP in cultured endothelial cells.
Am J Physiol Heart Circ Physiol
264:
H150-H156,
1993
27.
Kuchan, MJ,
and
Frangos JA.
Role of calcium and calmodulin in flow-induced nitric oxide production in endothelial cells.
Am J Physiol Cell Physiol
266:
C628-C636,
1994
28.
Kuchan, MJ,
Jo H,
and
Frangos JA.
Role of G proteins in shear stress-mediated nitric oxide production by endothelial cells.
Am J Physiol Cell Physiol
267:
C753-C758,
1994
29.
Leffler, CW,
Hessler JR,
and
Green RS.
The onset of breathing at birth stimulates pulmonary vascular prostacyclin synthesis.
Pediatr Res
18:
938-942,
1984[Abstract].
30.
Li, H,
Oehrlein SA,
Wallerath T,
Ihrig-Biedert I,
Wohlfart P,
Ulshofer T,
Jessen T,
Herget T,
Forstermann U,
and
Kleinert H.
Activation of protein kinase C-alpha and/or -epsilon enhances transcription of the human endothelial nitric oxide synthase gene.
Mol Pharmacol
53:
630-637,
1998
31.
Lin, K,
Hsu PP,
Chen BP,
Yuan S,
Usami S,
Shyy JY,
Li YS,
and
Chien S.
Molecular mechanism of endothelial growth arrest by laminar shear stress.
Proc Natl Acad Sci USA
97:
9385-9389,
2000
32.
Luscher, TF,
Yang Z,
Tschudi M,
Von Segesser L,
Stulz P,
Boulanger C,
Siebermann R,
Turnia M,
and
Buhler FR.
Interaction between endothelin-1 and endothelium-derived relaxing factor in human arteries and veins.
Circ Res
66:
1088-1094,
1990[Abstract].
33.
Moore, P,
Velvis H,
Fineman JR,
Soifer SJ,
and
Heymann MA.
EDRF inhibition attenuates the increase in pulmonary blood flow due to oxygen ventilation in fetal lambs.
J Appl Physiol
73:
2151-2157,
1992
34.
Morawietz, H,
Talanow R,
Szibor M,
Rueckschloss U,
Schubert A,
Bartling B,
Darmer D,
and
Holtz J.
Regulation of the endothelin system by shear stress in human endothelial cells.
J Physiol (Lond)
525:
761-770,
2000
35.
Morin, FC, III.
Ligating the ductus arteriosus before birth causes persistent pulmonary hypertension in the newborn lamb.
Pediatr Res
25:
245-250,
1989[Abstract].
36.
Nerem, R,
and
Girard P.
Haemodynamic influences on vascular endothelial biology.
Toxicol Pathol
18:
572-582,
1990[ISI][Medline].
37.
Nerem, RM,
Harrison DG,
Taylor WR,
and
Alexander RW.
Hemodynamics and vascular endothelial biology.
J Cardiovasc Pharmacol
21:
S6-S10,
1993[ISI][Medline].
38.
Nishida, K,
Harrison DG,
Navas JP,
Fisher AA,
Dockery SP,
Uematsu M,
Nerem RM,
Alexander RW,
and
Murphy TJ.
Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase.
J Clin Invest
90:
2092-2096,
1992[ISI][Medline].
39.
Noris, M,
Morigi M,
Donadelli R,
Aiello S,
Foppolo M,
Todeschini M,
Orisio S,
Remuzzi G,
and
Remuzzi A.
Nitric oxide synthesis by cultured endothelial cells is modulated by flow conditions.
Circ Res
76:
536-543,
1995
40.
North, AJ,
Star RA,
Brannon TS,
Ujiie K,
Wells LB,
Lowenstein CJ,
Snyder SH,
and
Shaul PW.
Nitric oxide synthase type I and type III gene expression are developmentally regulated in rat lung.
Am J Physiol Lung Cell Mol Physiol
266:
L635-L641,
1994
41.
Perreault, T,
and
De Marte J.
Maturational changes in endothelium-derived relaxations in newborn piglet pulmonary circulation.
Am J Physiol Heart Circ Physiol
264:
H302-H309,
1993
42.
Ranjan, V,
Xiao Z,
and
Diamond SL.
Constitutive NOS expression in cultured endothelial cells is elevated by fluid shear stress.
Am J Physiol Heart Circ Physiol
269:
H550-H555,
1995
43.
Resnick, N,
Collins T,
Atkinson W,
Bonthron D,
Dewey C, Jr,
and
Gimbrone M, Jr.
Platelet-derived growth factor B chain promoter contains a cis-acting fluid shear-stress-responsive element.
Proc Natl Acad Sci USA
90:
4591-4595,
1993[Abstract].
44.
Rosales, OR,
Isales C,
Nathanson M,
and
Sumpio BE.
Immunocytochemical expression and localization of protein kinase C in bovine aortic endothelial cells.
Biochem Biophys Res Commun
189:
40-46,
1992[ISI][Medline].
45.
Rudolph, AM.
Fetal and neonatal pulmonary circulation.
Annu Rev Physiol
41:
383-395,
1979[ISI][Medline].
46.
Sdogous, HP,
Bussolari SR,
and
Dewey CF.
Secondary flow and turbulence in a cone-and-plate device.
J Fluid Mech
138:
379-404,
1984[ISI].
47.
Shaul, PW,
Farrar MA,
and
Magness RR.
Pulmonary endothelial nitric oxide production is developmentally regulated in the fetus and newborn.
Am J Physiol Heart Circ Physiol
265:
H1056-H1063,
1993
48.
Sheehy, AM,
Phung YT,
Riemer KR,
and
Black SM.
Growth factor induction of nitric oxide synthase in rat pheochromocytoma cells.
Mol Brain Res
52:
71-77,
1997[ISI][Medline].
49.
Tiktinsky, MH,
Cummings JJ,
and
Morin FC, III
Acetylcholine increases pulmonary blood flow in intact fetuses via endothelium-dependent vasodilation.
Am J Physiol Heart Circ Physiol
262:
H406-H410,
1992
50.
Tiktinsky, MH,
and
Morin FC, III.
Increasing oxygen tension dilates fetal pulmonary circulation via endothelium-derived relaxing factor.
Am J Physiol Heart Circ Physiol
265:
H376-H380,
1993
51.
Traub, O,
Monia BP,
Dean NM,
and
Berk BC.
PKC- is required for mechano-sensitive activation of ERK1/2 in endothelial cells.
J Biol Chem
272:
31251-31257,
1997
52.
Uematsu, M,
Ohara Y,
Navas JP,
Nishida K,
Murphy TJ,
Alexander RW,
Nerem RM,
and
Harrison DG.
Regulation of EC nitric oxide synthase mRNA expression by shear stress.
Am J Physiol Cell Physiol
269:
C1371-C1378,
1995
53.
Vane, JR,
Anggard EE,
and
Botting RM.
Regulatory functions of the vascular endothelium.
N Engl J Med
323:
27-36,
1990[ISI][Medline].
54.
Zayek, M,
Wild L,
Roberts JD,
and
Morin FC.
Effect of nitric oxide on survival and lung injury in newborn lambs with persistent pulmonary hypertension.
J Pediatr
126:
947-952,
1993.
55.
Zellers, TM,
and
Vanhoutte PM.
Endothelium-dependent relaxations of piglet pulmonary arteries augment with maturation.
Pediatr Res
30:
176-180,
1991[Abstract].