From the
Endothelins (ETs) were initially characterized as potent
vasoactive peptides acting through at least two distinct receptors,
ET
Endothelins (ETs)
Both neonatal and adult cardiocytes show a
hypertrophic response to ET-1(10, 11, 12) , as
evidenced by increased protein synthesis and assembly of contractile
proteins into sarcomeric units. As well, ET-1 directly stimulates the ANP gene and increases secretion of the mature ANP hormone in vitro and in vivo(13) . A number of other
cardiac-specific genes have been shown to be activated by ET-1, namely
those encoding myosin heavy and light chains,
The physiological role of ETs
in the central nervous system remains unclear. The presence of ET-1 and
ET-3 immunoreactivity or mRNA has been established in multiple sites
including the cerebral cortex, the brainstem, the hypothalamus, the
basal ganglia, the cerebellum, and the spinal
cord(14, 15, 16, 17, 18) . Such
widespread distribution strongly suggests the possibility that ETs act
as neurotransmitters, neuromodulators, or neurohormones.
The effects
of ETs are mediated by specific high affinity cell surface receptors
(ETR), which are distributed not only in the vasculature but also in
the brain, kidney, heart, lung, adrenal cortex, uterus, and other
organs. At least two types of receptors (ET
In the present study, we have characterized a
model system of selective ET
P19 cells were obtained from the American Type Culture
Collection (ATCC, No. CRL 1825) or kindly donated by Dr. Michael
McBurney, Ottawa, Canada. Rat A10 thoracic aorta smooth muscle cells
were obtained from the ATCC (No. CRL 1476). Alpha minimum essential
medium and fetal bovine and newborn calf serum were from Life
Technologies, Inc. All-trans-retinoic acid was from Eastman
Kodak Co., dimethyl sulfoxide (Me
Binding experiments were performed
at room temperature for 4 h in a total volume of 0.2 ml of buffer A
containing 0.2% bovine serum albumin and 0.3% bacitracin with
15-20 µg of membrane protein from unaggregated and uninduced
cells, 4-8 µg from Me
Partial length cDNA fragments for the ET
Total RNA was isolated from the P19 cells by extraction with
guanidinium thiocyanate/acid phenol utilizing the commercially
available preparation RNAzol B (Tel-Test, Friendswood, TX)(31) .
Poly(A)
Fragments containing approximately 850 bp of the 5`-flanking
region and 90 bp of the first exon of the human ET
Expression plasmids were transfected into differentiated and
undifferentiated P19 cells by employing cationic liposomes and
serum-free conditions (Lipofectin and Opti-MEM respectively; Life
Technologies, Inc.) and following the protocols of the manufacturer.
Transfection conditions were optimized with the reporter plasmids
pSV-
Endothelins (ET-1, -2, and -3) were initially characterized
as a peptide family with marked effects on vascular
tone(1, 2) . Besides their potential role in
cardiovascular regulation, these peptides were also shown to elicit
mitogenesis and stimulate the synthesis of DNA in a broad range of
cardiovascular and noncardiovascular cells, such as vascular
endothelial and smooth muscle cells, cardiac myocytes, fibroblasts,
glomerular mesangium, and neural cells(38) .
The
physiological role of endothelins remains unclear. Expression of ET-1,
for instance, is very low in the plasma and tissues of normal adult
organisms but is induced to a very significant extent during extreme
pathophysiological conditions such as acute myocardial infarction,
cardiogenic and septic shock, pulmonary hypertension, and idiopathic
pulmonary fibrosis(39, 40, 41) . In contrast,
significant expression of ET-1 has been reported in normal embryonic
and fetal tissues raising the possibility that the endothelin family
may play a role in differentiation, growth, and
development(42) .
While ET
To test the hypothesis that cells at
various stages of development or differentiation would express ET
receptors differentially, we utilized the mouse embryonal carcinoma
cell line P19. In view of their antigenic and biochemical resemblance
to the cells of the inner cell mass of the blastocyst(44) ,
these cells have been widely used to study the events occurring during
the early development of the mammalian embryo. To the best of our
knowledge this is the first report on the predominant, and nearly
selective, expression of ET
In the classical receptor
binding experiments we demonstrated that the process of differentiation
was associated with a striking increase in the density of binding sites
for ETs in both the myocyte and the neural lineage. Competition with
the specific ET
Thus, upon differentiation into the two different lineages, the P19
cells expressed the predominant receptors observed in myocardium
(ET
Further evidence supporting the selective
pattern of receptor induction was provided by Northern blotting
analysis of P-19 mRNA. ET
Our reporter gene experiments with firefly
luciferase suggest that the differentiation of the P19 cells is
associated with selective transcriptional activation of the human ETR
genes. Thus, the differentiation of the P19 cells constitutes an
excellent model system to study the expression of ETR during
differentiation, in particular, and the regulation of ETR expression in
general. Ongoing studies utilizing this model system may allow us to
identify the cis- and trans-acting factors that control the selective
induction of these genes.
Furthermore, the striking level of
induction of endothelin receptors observed during the differentiation
of the P19 cells and the high affinity of the receptors (K
Further support for the role of endothelins and
their receptors in mammalian development has been provided by three
very recent reports on ET
Hence, endothelins and their receptors play a critical role in
specific aspects of mammalian differentiation and development. ET-1 and
its specific receptor ET
In view of the importance of the endothelin
system in differentiation of myocyte and neural tissues, a better
understanding of the transcriptional events which control the tissue
specific expression of ETR is essential for a broader comprehension
both of normal developmental events and the perturbations which produce
congenital abnormalities.
and ET
. Subsequently, their significant
growth- and hypertrophy-promoting properties in cardiac and other cells
were recognized. We investigated the expression of endothelin receptors
during differentiation of a pluripotential embryonal carcinoma cell
line (P19) to a cardiomyocyte or a neural lineage. These cells resemble
those of the inner cell mass of the blastocyst, and their
differentiation is believed to closely mimic critical events in early
embryogenesis. Differentiation of P19 to a cardiomyocyte lineage, by
aggregation and exposure to dimethyl sulfoxide resulted in induction of
ET
receptors as demonstrated by radioligand binding
studies, Northern blotting, and reporter gene analysis. Moreover, the
P19 differentiated to a cardiac lineage responded to ET-1 with a 3-fold
increase in the secretion of atrial natriuretic peptide. In contrast,
differentiation to a neural lineage, by aggregation and exposure to
retinoic acid, was associated with the induction of predominantly
ET
. Therefore, selective differentiation of the P19 led to
the differential expression of endothelin receptors in a pattern
consistent with that observed in normal myocardial and neural tissue.
The induction of endothelin receptors in a model system of early
embryogenesis provides strong support for the critical role of this
peptide/receptor family in differentiation and development. As well,
this model system is well suited for the study of the mechanisms
controlling endothelin receptor expression during differentiation.
(
)are a family of at
least three peptides (ET-1, -2, -3) with a variety of biological
activities(1, 2) . ET-1 is an extremely potent
constrictor of arterial and venous segments, and it also has a positive
inotropic effect in isolated myocardial
preparations(3, 4, 5) . In addition to its
cardiovascular actions, it contracts nonvascular smooth muscle cells
(bronchial, uterine, and gastrointestinal tract), it is a potent and
direct stimulator of synthesis of aldosterone and atrial natriuretic
peptide (ANP), and it modulates neural
transmission(6, 7, 8) . Moreover, ET-1 promotes
the growth of several cell types (myocardiocytes, vascular smooth
muscle, fibroblasts, mesangial, and neural cells), suggesting a role in
the regulation of cellular growth and tissue differentiation, as well
as in pathological proliferative processes such as atherosclerosis and
tumor growth(9) .
-actin, and
troponin-T. Therefore, ET-1 has been proposed as an important paracrine
mediator in the regulation of myocardial growth and
development(11, 12) .
and
ET
) have been characterized and cloned thus
far(19, 20) . They differ in their distribution and in
their selectivity for ET isoforms. Both have seven putative
transmembrane domains similar to other members of the G-protein-coupled
receptor family. The ET-1 (and ET-2) selective ET
type is
the predominant receptor of cardiac myocytes and smooth muscle cells.
The ET
receptor exhibits equal affinity for the three
isoforms and is the predominant, if not exclusive, receptor in neural
tissue. Both types are found in various proportions in lung and renal
medulla(21) .
or ET
receptor
induction during differentiation of P19, a pluripotential embryonal
carcinoma cell line (22-24), along a cardiac myocyte or a neural
lineage, respectively. In view of the fact that ETs have growth- and
hypertrophy-promoting activity, the demonstration of endothelin
receptors in cells that resemble those of the early embryo would
support a role of endothelins and their receptors in growth and
differentiation.
Cell Culture and Differentiation
SO) was from Sigma.
Differentiation of the P19 cells to a myocyte lineage or a neural
lineage was carried out by aggregation and exposure to 1%
Me
SO (v/v) or 0.3 µM retinoic acid,
respectively, following a previously described procedure(25) .
Radioligand Binding Experiments
Intact Cells
The medium was removed from the
cells growing in 24-well plates, replaced by 1 ml of sterile fresh
Ham's F-12 medium containing 0.2% bovine serum albumin and 0.3%
bacitracin, and the cells were allowed to equilibrate for 1 h at 37
°C. The binding of I-ET-1 or
I-ET-3
(both at specific activity of 2000 Ci/mmol, Amersham Corp.) was then
performed in fresh medium (0.2 ml/well) at 37 °C. In competition
binding experiments, cells were incubated with 50 pM radioligand and various concentrations of unlabeled competitors
(ET-1 and ET-3 from Peninsula Laboratories, Belmont, CA, BQ123 from
Peptides International, Louisville, KY). The reaction was stopped by
aspiration of the media. The cells were then washed sequentially with 1
ml of ice-cold 0.1% bovine serum albumin in phosphate-buffered saline
(PBS) and 1 ml of PBS. After digestion in 2 N NaOH,
radioactivity and protein concentration of the digest were measured by
an automated gamma-counter and the Bradford assay (Bio-Rad),
respectively. Nonspecific binding defined as cell-associated
radioactivity in the presence of 0.2 µM unlabeled ET-1 did
not exceed 10% of total radioactivity. Degradation of radioligand was
monitored routinely by precipitation of the medium with 12%
trichloroacetic acid. It did not exceed 4%. Similar results were found
in representative experiments with high performance liquid
chromatography analysis of cell-exposed media.
Membranes
Membranes were prepared from cells grown
on 15-cm cell culture dishes. The cells were washed with ice-cold PBS,
scraped with a rubber policeman, suspended in 0.3 M sucrose
with 5 mM HEPES (pH 7.4), and homogenized on ice with a
Polytron (Brinkman). The homogenate was centrifuged at 1,000 g for 10 min at 4 °C, and the supernatant was collected
and centrifuged at 35,000
g for 30 min at 4 °C.
The pellet was resuspended in buffer A (in mM: 124 NaCl, 5
KCl, 1 CaCl
, 0.4 MgSO
, 1
NaH
PO
, 50 HEPES, pH 7.4), aliquoted, and stored
at -80 °C for usually not more than 2 weeks. In preliminary
experiments, no change in binding parameters was observed under these
conditions for at least 2 months.
SO-induced cells, and
3-6 µg from RA-induced cells. The reaction was stopped by
addition of 1 ml of ice-cold PBS and rapid centrifugation at 12,000
g for 3 min. In competition experiments,
concentrations of
I-ET-1 and of unlabeled competitors
were as described with intact cells. In saturation experiments,
membranes were incubated with increasing concentrations (18 to 2,000
pM) of radioiodinated ET-1 or ET-3. Nonspecific binding of
I-ET-1 was assessed as above, while that of
I-ET-3 was determined in the presence of 1 µM of unlabeled ET-3. Kinetic parameters (B
, K
) were obtained by an iterative
nonlinear least-square regression analysis of bound and free
radioactivity data.
Determination of Secreted Peptides
Measurement of ANP
Measurement of ANP in the
media was done by a specific radioimmunoassay of SepPak-extracted media
as described previously(26) . Detection limit of the assay was
0.4 pg/tube, and the intra- and interassay coefficient of variation was
7 and 11%, respectively.
Immunoreactive ET-1
Immunoreactive ET-1 was
determined with the radioimmunoassay established in our laboratory (27) after extraction on SepPak-C18 cartridges (Waters
Millipore, Missisauga, Ontario). After loading the media on the
cartridges and washing with 10 ml of HO, peptides were
eluted with 100% methanol with 75% recovery. The detection limit was
0.1 pg/tube, and the intra- and interassay coefficient of variation was
9 and 12%, respectively. Cross-reactivity of ET-3 and big ET-1 with the
rabbit anti-ET-1 serum (Peninsula Laboratories, RAS 6901) was 5 and
10%, respectively.
Cloning of the cDNAs for Mouse Endothelin Receptors
and
ET
receptors were obtained by reverse
transcription-polymerase chain reaction (PCR) from total RNA from P19
cells differentiated with Me
SO or RA, respectively.
Oligonucleotide primers were designed, based on the rat ETR sequences
(20, 28), which are complementary to highly conserved regions in the
first and third transmembrane domains. The sequence of the primers was
as follows: 1) ET
sense, 5`-TTTTCATCGTGGGAATGGTGGG-3`;
ET
antisense, 5`-GACTTCTGCAAAAAGGGGAACA-3`; and 2) ET
sense, 5`-GTATCATGCCTCGTGTTCGTG-3`; ET
antisense,
5`CGACTCCAAGAAGCAACAGCT-3`. Amplification of human placental cDNA,
bovine aorta and pulmonary artery, and rat brain and kidney cDNA
yielded fragments of the predicted size (234 bp for ET
and
300 bp for ET
). Subsequently, the primers were utilized in
reverse transcription-PCR of the P19 RNA. The conditions for reverse
transcription are as published elsewhere(29) . Amplification was
carried out for 40 cycles as follows: denaturing at 94 °C for 1
min, annealing at 55 °C for 1 min, extension at 72 °C for 1 min
with increments of 2 s/cycle, followed by a 10-min extension after the
final cycle. After chloroform extraction, the reaction was analyzed in
a 2.5% agarose gel revealing products of the same size as those of the
other species. The PCR products were then ligated into the PCR II
vector (Invitrogen; San Diego, CA) and sequenced by the dideoxy method.
Inserts were identified as endothelin receptors by sequence analysis of
the GenBank
data base using the BLAST program(30) . The
nucleotide sequences in the amplified region were highly homologous to
the respective human receptors. For subsequent experiments, fragments
were excised by EcoRI digestion, purified by preparative
electrophoresis and the QIAEX method (QIAGEN, Chatsworth, CA), and
radiolabeled with [
-
P]dCTP (Amersham Corp.)
utilizing T7 DNA polymerase and the random primer method with a kit
from Pharmacia Biotech Inc. Specific activities of 1-3
10
dpm/µg of DNA were achieved.
Analysis of Endothelin Receptor mRNA Levels in
Differentiated and Undifferentiated P19 Cells
RNA was isolated by the Poly(A) tract system
(Promega) and fractionated by electrophoresis on a denaturing
formaldehyde agarose gel. Capillary transfer to Hybond N membranes
(Amersham Corp.) was performed subsequently. RNA was cross-linked to
the membranes with an ultraviolet cross-linker (Fisher Scientific,
model UVXL) utilizing the preprogrammed optimal cross-link setting. For
hybridization and posthybridization washes, the membranes were
processed as described previously(32) . ETR probes were added in
concentrations of 10
dpm/ml. Autoradiograms of the
membranes were obtained with exposures of 1-3 days utilizing
double intensifying screens and Kodak XAR film.
Isolation of Promoter Fragments of ETR Genes and
Preparation of Luciferase Expression Plasmids
gene (33) and 1.1 kilobase pairs of the flanking region and 250 bp of
the first exon of human ET
gene (34) were
obtained by PCR of human genomic DNA as described
previously(35) . The primers employed were for ET
, 5`-GTCGGTACCGGATCCTCCAGCCCCTGCTACAT-3` (sense) and
5`-GCTCTCGAGTGTCCTCCCCGTCTCCTCCCAAA-3` (antisense); and for
ET
, 5`-GCTGGTACCGTGCGTGATAACTTGCCCTTG-3` (sense) and
5`-GCTCTCGAGTAGTGGGTGGCGTCATTATCT-3` (antisense). A KpnI site
was included in both sense primers, whereas XhoI sites were
incorporated into both antisense primers. After digestion with the
appropriate enzymes to generate the restriction sites, the fragments
were inserted into the pGL2 enhancer vector (Promega). Sequences were
confirmed by dideoxy sequencing in the double-stranded plasmids.
(
)Large scale preparations of expression plasmids for
transfection were performed by the method of QIAGEN Inc.
Transfection of P19 Cells
-galactosidase and pGL2-control (Promega) in which the
expression of the
-galactosidase and firefly luciferase genes,
respectively, are regulated by the SV40 early promoter and enhancer.
Optimal transfection conditions for a 35-mm dish were 10 µg of
Lipofectin, 1-5 µg of plasmid DNA, and 6 h of incubation in
serum-free conditions. The peak levels of transient expression of the
reporter genes was observed at 48 h (not shown). These conditions were
utilized in all subsequent experiments with the ETR promoter
constructs.
-Galactosidase activity was assayed by a colorimetric
assay(36) . Luciferase was quantified in a Berthold AG
luminometer according to a previously described protocol(37) .
Binding of Radioiodinated Endothelins
The intact
cells growing in monolayers and not subject to aggregation exhibited a
barely detectable binding of 50 pMI-ET-1. After
aggregation, and more markedly after induction with RA or with
Me
SO, there was a clear increase of binding. In the
competition experiments (Fig. 1), unlabeled ET-1 readily
displaced
I-ET-1 in both RA- and Me
SO-induced
cells with similarly high affinity (IC
of 80 and 110
pM, respectively). While the linear Scatchard plots derived
from these data suggested an apparent homogeneity of the binding sites,
the affinity profile of BQ123 revealed its heterogeneous nature. This
ET
-selective antagonist was a very weak competitor in
RA-induced cells (IC
almost 100 µM), while it
potently displaced the radioligand in Me
SO-induced cells
with IC
1000 times lower. Hence in
Me
SO-induced cells, the majority of ET-1 binding was due to
ET
receptors, while the RA-induced cells expressed mostly
ET
receptor. The similarity of the affinity profiles of
BQ123 and ET-3 obtained in rat aortic smooth muscle cells (not shown),
known to express almost exclusively the ET
receptor type
with the profile found in Me
SO-induced cells, supported
this notion.
Figure 1:
Competition with binding of I-ET-1 by cold ET-1 (circles), ET-3 (squares), and BQ123 (triangles) in
Me
SO-induced (full line) and RA-induced (dashed line) P19 cells.
Membranes were then obtained from five different
preparations of each of the following, nonaggregated, aggregated,
MeSO-induced, and RA-induced cells, and were used to extend
the competition binding studies, as well as to examine the density of
binding sites (B
) both with
I-ET1
and
I-ET3. The increase in B
of
ET-1 binding as assessed by Scatchard analysis of competition binding
data (Fig. 2) was 20-, 100-, and 180-fold in aggregated,
Me
SO-induced, and RA-induced cells, respectively, with K
values in the range of 40 to 150
pM. When the time course of binding of
I-ET-1
and
I-ET-3 was compared (Fig. 3), RA-induced cells
bound both radioligands almost equally, while Me
SO-induced
cells bound only
I-ET-1. Affinity profiling with ET-1,
ET-3, and BQ123 gave similar results as those obtained in the intact
cells, thus confirming the prevalence of ET
-type receptors
in Me
SO-treated cells and that of ET
type in
RA-induced cells. Saturation binding experiments both with
I-ET-1 and
I-ET-3 also showed that the
majority of ET-1-binding sites (60-90% in various preparations)
in RA-induced cells belong to ET
type. In contrast,
Me
SO-derived membranes bound almost exclusively
radiolabeled ET-1, again compatible with a strikingly prevailing
presence of ET
type receptors (Fig. 4).
Figure 2:
Scatchard plot of competition binding data
obtained with I-ET-1 as radioligand and cold ET-1 as
competitor in membranes from control (nonaggregated, diamonds, and inset), aggregated (triangles), Me
SO-induced (squares), and
RA-induced (circles) P19 cells.
Figure 3:
Time course of binding of I-ET-1 (thick line) and
I-ET-3 (thin line) in membranes obtained from
Me
SO-induced (squares), and RA-induced (circles) cells. The concentration of both ligands was 50
pM. The experiment was repeated three times; a representative
result is presented.
Figure 4:
Binding of increasing concentrations of I-ET-1 (circles, squares) and
-I-ET-3 (triangles), Me
SO-induced (squares), and RA-induced (circles, triangles) P19 cells. Binding of
I-ET-3 in
Me
SO-induced cells was barely
detectable.
Secretion of ET-1 and ANP by P19 Cells
To
ascertain whether the apparent density of binding sites could be
affected by prior receptor occupancy by ET-1 secreted by the cells,
immunoreactive ET-1 levels were measured in the conditioned media (Fig. 5A). After a 24-h incubation, the highest
secretion levels were found in undifferentiated cells, but never
exceeded 5 pM, corresponding to secretion rate of 90-100
pg/mg of cell protein/24 h. At this concentration, ET-1 inhibited
binding of radioligand by <5% in competition studies (Fig. 1).
Hence, the effect of the endogenous peptide on the results of binding
experiments was negligible even in the cells with the highest secretory
activity, i.e. undifferentiated P19. Aggregation alone or
associated with MeSO induction markedly decreased ET-1
secretion. The cells induced with RA secreted only 10% of the amount
produced by undifferentiated cells. To evaluate the functionality of
the receptor induced during differentiation, we also measured secretion
of ANP and its response to ET-1 added to the media (Fig. 5B). Negligible secretion of ANP in
undifferentiated cells was not affected by addition of ET-1.
Aggregation alone resulted in a slight increase in ANP production, but
again without any response to ET-1. In contrast, the highest secretory
rates were observed in the cells differentiated to a myocyte lineage
and, more importantly, only these cells exhibited a marked (3-fold, p < 0.01) increase in ANP secretion in the presence of
ET-1. This response, characteristic for cardiac myocytes in vivo and in vitro, suggests that ET
receptors
induced in these cells were functional and coupled to the appropriate
signal transduction mechanisms.
Figure 5:
A, secretion of ET-1 in P19 cells. Mean
± S.E. of triplicate measurements is presented. *, significantly
different from control (nonaggregated) P19 cells. B, secretion
of ANP in P19 cells in the absence and presence of ET-1
(10M). Mean ± S.E. of triplicate
measurement is presented. *, significantly higher than in the absence
of ET-1.
Expression of ETR mRNA during Differentiation
The
levels of ETR mRNA determined by Northern blotting were insignificant
in the exponentially growing cells and in cells subjected to the
aggregation protocol without inducing agents. In contrast, selective
increases in ETR mRNA were detected in the differentiated cells,
specifically a marked predominance of ET mRNA after RA
induction and exclusively ET
after induction with
Me
SO (Fig. 6). No difference was observed in the mRNA
levels of the constitutively expressed glyceraldehyde-3-phosphate
dehydrogenase between undifferentiated and differentiated P19 cells.
These findings are in complete agreement with those of the receptor
binding studies and support our conclusion that competition by secreted
ET-1 in the media is not the explanation for the negligible binding of
ET-1 to the undifferentiated cells.
Figure 6:
Northern blotting of mRNA from P19 cells
(10 µg/lane): 1, monolayers of exponentially-growing
undifferentiated P19 cells; 2, P19 aggregated in the absence
of inducers; 3, P19 differentiated to a neural lineage; 4, P19 differentiated to a cardiomyocyte
lineage.
Luciferase Expression in Differentiated P19 Cells
Directed by ETR Promoters
To evaluate further the mechanisms
responsible for the expression of the ETR genes during differentiation
of the P19 cells to a myocyte or a neural lineage, we constructed
expression plasmids in which the expression of the firefly luciferase
gene was driven by fragments from the human ET or ET
promoters. The major putative binding sites for transcription
factors present in the ET
promoter fragment are: a CArG
box, an inverted MyoD-E2A binding site, an SP-1 binding site, and four
GATA motifs. The ET
promoter fragment contains the
following putative binding sites, a GATA motif, four ``E
boxes,'' an acute phase reactant regulatory element (APRRE), and
an inverted APRRE. The human ET
promoter was able to direct
the expression of luciferase in the Me
SO-induced cells and
to a much lesser degree in the RA-induced cells. In contrast, the human
ET
construct directed the expression of luciferase only in
the RA-induced cells (Fig. 7). Minimal luciferase activity was
observed with all constructs in the undifferentiated cells. These
results were in complete agreement with the mRNA and binding data and
are interpreted as indicating that, in least in part, the induction of
expression of the ETR genes during differentiation of the P19 cells is
transcriptionally regulated. Mock transfected P19 cells of all types,
as well as cells transfected with promoterless luciferase plasmids,
expressed levels of luciferase which were, in most instances, not
significantly different from the background of the luminometer.
Figure 7:
Luciferase activity (RLU) in
differentiated P19 cells (clear bars, cardiomyocyte lineage; shaded bars, neural lineage) transfected with ETR promoter
constructs (hETAR, ET promoter-luciferase; hETBR, ET
promoter-luciferase). *p <
0.01 compared to the other bars of the same shading.
,
Me
SO;
, RA.
appears to be the only
receptor expressed in neural tissue, both ET
and ET
have been observed in the heart(21) . In situ hybridization analysis of receptor mRNA revealed the expression of
ET
- and ET
-specific transcripts (with a
predominance of the former) both in the atria and
ventricles(21) . Binding experiments with a selective ET
antagonist (BQ123) showed, as well, that the majority of binding
sites on cardiac myocytes belong to the ET
class(43) .
and ET
receptors
during differentiation of a pluripotential cell line toward a cardiac
myocyte or a neural lineage, respectively.
antagonist BQ123 established that the vast
majority of the receptors expressed during differentiation of the cells
in the presence of Me
SO (myocyte lineage) are of the
ET
type. In contrast, the binding of labeled ET-3 and
competition with unlabeled ET-3 determined that there was a marked
predominance of ET
receptors in the cells induced to
differentiate to a neural lineage with retinoic acid. In these cells a
second type of receptor, presumably ET
, was also present
but accounted for no more than 10-40% of the binding sites.
) or in neural tissue (ET
)(21) .
Moreover, differentiation of P19 cells toward a myocyte lineage
resulted in the induction of ANP secretion, which was increased in
response to ET-1, an ET
-mediated phenomenon well known to
occur in normal cardiomyocytes(12, 45) . ET-1-responsive
secretion of ANP does not by itself establish the cardiac nature of the
Me
SO-induced cells as ANP is produced in other tissues.
However, their response to ET-1 suggests that the ET
induced are functional and coupled to appropriate signal
transduction mechanisms.
mRNA was observed only in the
RA-induced cells and was indeed the most abundant of the ETR mRNAs
observed in these studies. Visualization of ET
mRNA, in
contrast, required longer exposure times in both the RA- and
Me
SO-induced cells. In the latter only ET
mRNA
was detectable.
values of 40-150 pM)
strongly support a physiological role for the ETR in these embryonal
cells. The importance of ETR in embryogenesis has been underscored by
recent studies of targeted disruption of the ET-1 and ET
genes. Mice homozygous for the disruption of either
gene exhibited severe developmental abnormalities of structures derived
from the first pharyngeal arch and died shortly after birth of
respiratory failure(46, 47) . Moreover, following ET
disruption a high incidence of abnormal cardiac
development was observed, with at least 50% of animals demonstrating
membranous ventricular septal defects.
(
)
and ET-3. The human ET
gene was identified as the recessive susceptibility
locus for Hirschprung's disease in chromosome 13q22 (48). An
absence of enteric ganglia in the distal colon and a failure of
innervation in the gastrointestinal tract are characteristic of this
disease. Moreover, in patients with Hirschprung's disease a G
T mutation was identified in exon 4 of the ET
gene resulting in the substitution of the highly conserved
Trp-276 residue in the fifth transmembrane domain of the receptor with
a Cys residue. This mutation produces a receptor with an impairment of
ligand-induced responses. Additionally, the targeted (gene knockout)
and natural (piebald-lethal) mutations of mouse ET
both
produce aganglionic megacolon, analogous to the human
Hirschprung's disease, and abnormalities in epidermal melanocytes
leading to a spotted coat color(49) . Finally, the targeted
disruption of the mouse ET-3 leads to a similar, although
slightly less severe, phenotype of megacolon and color
spotting(50) . A natural recessive mutation that results in the
same developmental defects in mice, lethal spotting, is associated with
a point mutation of the ET-3 gene, which replaces the Arg
residue at the C terminus of big ET-3, an inactive precursor, with a
Trp residue and prevents the generation of mature ET-3(50) .
are important in the
differentiation of tissues derived from the first branchial arch,
particularly those in which cell lineages of the cephalic neural crest
play an essential role. As well, ET-3/ET
interactions
contribute to the normal development of two additional neural crest
lineages, the vagal neural crest-derived enteric neurons and the trunk
neural crest-derived epidermal melanocytes. Our studies demonstrate the
selective induction of ETR in a process believed to mimic,
morphologically and biochemically, some of the critical events of early
embryogenesis. Thus, the present report provides a model system well
suited for the study of the cellular mechanisms that control the
selective expression of ETR during differentiation into two highly
relevant cell lineages.
/EMBL Data Bank with accession numbers
ET
, D11144, and ET
, D13162.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.