(Hypertension. 1998;32:917-922.)
© 1998 American Heart Association, Inc.
Scientific Contributions |
Autocrine Role for the Endothelin-B Receptor in the Secretion of Adrenomedullin
From the Cardiorenal Research Laboratory, Division of Cardiovascular Diseases, Mayo Clinic and Foundation, Rochester, Minn.
Correspondence to Michihisa Jougasaki, MD, PhD, Cardiorenal Research Laboratory, 915 Guggenheim, Mayo Clinic and Foundation, 200 First St SW, Rochester, MN 55905. E-mail jougasaki{at}mayo.edu
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Abstract—Adrenomedullin, originally discovered in human pheochromocytoma, is a vasodilating and natriuretic peptide of vascular endothelial and smooth muscle cell origin. Although endothelin-1 (ET-1) has been implicated as a vasoconstricting and growth-promoting peptide of endothelial origin, it may more importantly function as an autocrine factor and release vasodilatory substances such as nitric oxide by mechanisms linked to the endothelin-B (ETB) receptor subtype. The present study was designed to establish that the ETB receptor stimulates the secretion of adrenomedullin from cultured canine aortic endothelial cells. We first sought to determine the presence and production of adrenomedullin in canine aortic endothelial cells using immunohistochemistry and Northern blot analysis, which revealed that adrenomedullin immunoreactivity and adrenomedullin mRNA were present in canine aortic endothelial cells. Second, adrenomedullin was time-dependently secreted from canine aortic endothelial cells, with a secretion rate of 15.7±1.5 pg/105 cells per 24 hours. Furthermore, immunohistochemistry revealed the presence of the ETB receptor in canine aortic endothelial cells, and ETB receptor stimulation by sarafotoxin S6c increased adrenomedullin production and secretion from canine aortic endothelial cells. Such actions were blocked with the ETB receptor antagonist IRL-2500 but not with ETA receptor antagonist FR-139317. These studies are the first to report an additional autocrine role of the ETB receptor in the release of vasodilating and natriuretic peptide adrenomedullin, and they suggest another important vasoactive system regulated by the ET receptor subtype.
Key Words: endothelium • hormones • immunohistochemistry • peptides • radioimmunoassay
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Introduction |
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Adrenomedullin (ADM) is a 52–amino acid vasodilating and natriuretic peptide that possesses an intramolecular disulfide bond and C-terminal amide structure.1 2 Its biological actions involve activation of the second messenger cAMP.3 The presence of specific ADM receptors on vascular smooth muscle cells,3 along with studies that have reported the synthesis and secretion of ADM in vascular endothelial cells4 and smooth muscle cells,5 supports the concept that ADM may play an important autocrine and/or paracrine role in the regulation of vascular tone. Furthermore, ADM has been detected in normal human plasma, and its circulating concentrations are reported to be increased in patients with hypertension,6 chronic renal failure,6 and congestive heart failure.7 8 To date, however, control of ADM secretion has not been well characterized.
The vasoconstricting and mitogenic peptide endothelin-1 (ET-1)9 is released from vascular endothelial cells under normal and pathophysiological conditions and plays an important autocrine and/or paracrine role in the regulation of vascular tone. ET-1 exerts its biological actions through at least 2 receptor subtypes, the endothelin-A (ETA) and endothelin-B (ETB) receptors.10 11 While the ETA receptor may mediate vasoconstriction under pathophysiological conditions, the ETB receptor, which is localized to the endothelial and smooth muscle cells, may function under physiological conditions to mediate vasodilatation via release of potent vasodilators that include nitric oxide (NO) and prostacyclin (PGI2).12 13
Although both ET-1 and ADM are secreted from the vascular endothelial cells, little is known about the relationship between ET-1 and ADM. In vitro studies by Sugo et al14 have demonstrated that ET-1 stimulates ADM secretion from cultured vascular smooth muscle cells. To date, however, no in vitro studies have investigated the role of the ETB receptor in the release of ADM in vascular endothelial cells. We hypothesized that activation of the ETB receptor increases ADM secretion from vascular endothelial cells. To test this hypothesis, we investigated the presence and production of ADM in cultured canine aortic endothelial cells (CAECs) and defined the role of the ETB receptor by determining its presence in CAECs and by characterizing ADM production and secretion in response to the ETB receptor agonist sarafotoxin S6c. Last, we assessed whether ETB receptor blockade would attenuate such actions.
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Methods |
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Cell Culture
CAECs were prepared in a similar fashion as described previously.15 Briefly, endothelial cells were isolated from male mongrel dogs by scraping the intimal surface of the aorta with a scalpel and were cloned and maintained in Medium 199 (Biowhittaker) supplemented with 10% fetal calf serum at 37°C and 5% CO2 with 95% O2 in a humidified atmosphere. CAECs were identified by active production of ET-1, positive immunostaining with monoclonal anti-von Willebrand factor antibody, and negative immunostaining for
-smooth muscle cell actin. At
confluence, CAECs appeared as typical "cobblestone"-patterned
monolayers. CAECs in the fourth to eighth passage were used in the
present experiments.
Immunohistochemistry for ADM
For immunohistochemical analysis, cultured CAECs were
immediately fixed with 10% buffered formalin for 20 minutes. The cells
were washed 3 times with PBS. To block the activity of
endogenous peroxidase, the cells were incubated with 0.6%
hydrogen peroxide in methanol for 20 minutes at room temperature. After
washing, they were subsequently incubated with 5% goat serum (Dako)
for 10 minutes at room temperature to reduce nonspecific background
staining, and they were then incubated with rabbit polyclonal anti-ADM
antibody (Peninsula) at a dilution of 1:100 in humidified chambers for
24 hours at room temperature. The antibody to ADM showed no
cross-reactivities with other known endothelial
cell–derived vasoactive peptides, such as ET-1 and C-type
natriuretic peptide. The cells were then incubated for 30
minutes with second antibody–horseradish peroxidase conjugate (Tago)
at a dilution of 1:100. The final reaction was achieved by incubating
the cells with freshly prepared reagent containing
3-amino-9-ethylcarbazole (Sigma Chemical Co) dissolved in
dimethylformamide and sodium acetate. The cells were counterstained
with hematoxylin and reviewed with an Olympus microscope. Adsorption
tests were performed to examine the immunohistochemical specificity of
the reaction between anti-ADM antibody and cells. The anti-ADM antibody
was preincubated with 10-6 mol/L of human
ADM(1-52) (Phoenix Pharmaceuticals Inc) overnight. After
centrifugation at 4500 rpm for 10 minutes, the
supernatant was used instead of primary antibody. The specificity was
further confirmed by substitution of nonimmune rabbit serum (Dako) or
PBS for primary antiserum.
Immunohistochemistry for ETB Receptor
Fixation of CAECs and blockage of endogenous
peroxidase activity were achieved as described above. After blocking
the activity of endogenous peroxidase, CAECs were incubated
with 5% horse serum (Vector Laboratories) for 10 minutes at room
temperature to reduce nonspecific background staining and were then
incubated with sheep polyclonal anti-ETB antibody
(AbProbe International) at a dilution of 1:300 in humidified chambers
for 24 hours at room temperature. The antibody to the
ETB receptor showed no cross-reactivities with
other known endothelial cell–derived peptides. The
cells were then incubated for 30 minutes with biotinylated anti-sheep
immunoglobulin (Vector Laboratories) at a dilution of 1:100. After
being washed with PBS, the cells were incubated for 10 minutes with
streptavidin (Dako) at a dilution of 1:300. The final reaction with
3-amino-9-ethylcarbazole and counterstaining with hematoxylin were
performed as described above. The specificity of the
immunostaining was confirmed by substitution of
nonimmune horse serum (Vector Laboratories) or PBS for primary
antibody.
Pharmacological Treatment
After being washed 3 times with PBS, confluent monolayer CAECs
were preincubated with protein-free medium for 2 hours. Sarafotoxin S6c
(10-9 to 10-6 mol/L,
Sigma) was added with and without either the ETA
receptor antagonist FR-139317 (10-6
mol/L, Abbott Laboratories) or the ETB receptor
antagonist IRL-2500 (10-6 mol/L,
Phoenix Pharmaceuticals Inc) and incubated for the indicated time. To
determine the percentage of viable cells, the
endothelial cells were stained with a 0.25% solution
of trypan blue after incubation with and without drugs for 24
hours.
Radioimmunoassay for ADM
After incubation for the indicated time, the culture medium was
removed and immediately acidified with acetic acid (final
concentration, 1 mol/L), heated at 100°C for 10 minutes to
inactivate intrinsic protease, and stored at -20°C until
assay. Culture medium (1 mL) was extracted on C-18 Bond Elute
cartridges and eluted with 75% methanol containing 1% trifluoroacetic
acid. Concentrated eluates were then assayed using a specific and
sensitive radioimmunoassay for ADM(1-52) (Phoenix Pharmaceuticals Inc)
as previously described.7 8 Minimal detectable
concentration for the assay is 1 pg per tube, and the half-maximal
inhibition dose of radioiodinated ligand binding by ADM is
20 pg per tube. Recovery is 72%, and intra-assay and interassay
variations are 10% and 12%, respectively.
Radioimmunoassay for ET-1
The measurement of ET-1 in the culture medium was performed
using a specific and sensitive radioimmunoassay for ET-1 as previously
reported.16 Culture medium (1 mL) was extracted
on C-8 Bond Elute cartridges and eluted with 95% methanol
containing 1% trifluoroacetic acid. Concentrated eluates were then
assayed. Minimal detectable concentration for the assay is 0.5 pg per
tube, and the half-maximal inhibition dose of
radioiodinated ligand binding by ET-1 is 10 pg per tube.
Recovery is 71%, and intra-assay and interassay variations are 9% and
5%, respectively.
Isolation of mRNA
Isolation of mRNA from canine adrenal glands was performed using
the Micro-FastTrack kit (Invitrogen) following the manufacturer's
instructions. In brief, the adrenal glands were initially lysed in
detergent-based buffer containing RNAse/Protein Degrader, incubated at
45°C, and applied directly to oligo(dT) cellulose for adsorption.
DNA, degraded proteins, and cell debris were washed from the resin with
a high-salt buffer. Nonpolyadenylated RNA was washed off with a
low-salt buffer, and the polyA+ RNA was then
eluted in the absence of salt. Purity and quality of the RNA was
assessed by reading optical densities at 260 and 280 nm and by
electrophoresis in 1.2% agarose gel.
Reverse Transcription–Polymerase Chain Reaction
The cDNA sequence for canine ADM has not been reported. To
obtain a specific probe for canine ADM, reverse
transcription–polymerase chain reaction (RT-PCR) was performed using
primers that are highly conserved among species. The cDNA was prepared
from 1 µg mRNA using oligo(dT) primer and Moloney murine leukemia
virus reverse transcriptase (Perkin-Elmer). The
oligonucleotide primers were synthesized using a
Applied Biosystems 394 DNA/RNA synthesizer. For canine ADM
fragment amplification, the following porcine primers were selected
from the pub- lished sequence17: sense
(porcine ADM 91-113), 5'-CGAAAGAAATGGAATAAGTGGGC-3'; antisense (porcine
ADM 364-383), 5'-GTGAACTGGTAGATCTGGTG-3', yielding a predicted
product of 293 bp. A Perkin-Elmer 2400 Thermocycler was used to
amplify the samples. PCR cycling parameters were as
follows: initial denaturation at 94°C for 3 minutes followed by
cycles of denaturation at 94°C for 1 minute, annealing at 55°C for
1 minute, and extension at 72°C for 1 minute. Thirty-five cycles were
used with a final extension time of 7 minutes. The PCR products
were cloned into a TA cloning vector (Invitrogen) and sequenced by ABI
Prism dideoxy chain termination method using Applied Biosystems 377
Automated DNA Sequencer. The nucleotide sequence is
reported in Figure 1
. The clone contained
a 293-bp fragment, which was considered to encode the deduced structure
of canine ADM (GenBank accession number AF045773).
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Northern Blot Analysis
For Northern blot analysis, 4 µg mRNA from the cell
extracts was loaded on a 1.2% agarose formaldehyde gel and
electrophoresed for 3 hours at 70 V. The gel was transferred downward
(Turboblotter, Schleicher & Schuell) onto a nylon membrane (Maximum
Strength Nytran Membrane, Schleicher & Schuell) overnight. An
EcoRI DNA restriction fragment containing 293 bp gene for
canine ADM was labeled with 32P-dCTP by a
random-priming labeling kit (Megaprime DNA labeling system, Amersham)
and purified using G-50 NICK Spin Columns (Pharmacia Biotech). The
membranes were then washed in 2x SSC, 0.1% SDS at 22°C for 15
minutes, then in 0.2x SSC, 0.1% SDS at 22°C for 15 minutes, and
0.2x SSC, 0.1% SDS at 55°C for 20 minutes.
Autoradiography was carried out with Kodak x-ray film
at -80°C overnight. To control for loading conditions and mRNA
transfer onto membranes, blots were rehybridized with a GAPDH
probe.
Statistical Analysis
Results of quantitative studies are expressed as mean±SEM.
Statistical comparisons were performed using ANOVA for repeated
measures followed by Fisher's least significant difference test when
appropriate. Comparisons between groups were performed using Student's
unpaired t test. Statistical significance was accepted at a
value of P<0.05.
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Results |
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Immunohistochemical Stainings for ADM and ETB Receptor in CAECs
Positive immunostaining for ADM was observed in the cultured CAECs. Representative immunohistochemical staining for ADM in CAECs is illustrated in Figure 2
. ADM immunoreactivity was observed
within the cells and distributed widely in the peripheral
cytoplasm. There was also ADM immunoreactivity in the perinuclear
regions. The cells treated with preabsorbed antiserum, nonimmune rabbit
serum, or PBS instead of primary antibody demonstrated little or no ADM
immunoreactivity.
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Positive immunostaining for the
ETB receptor was also detected in the cultured
CAECs. Representative immunohistochemical staining for
the ETB receptor in CAECs is illustrated in
Figure 3. The distribution of
ETB receptor immunoreactivity was similar to that
of ADM immunoreactivity. The cells treated with nonimmune horse serum
or PBS instead of primary antibody demonstrated no
immunoreactivity.
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Basal and Stimulated ADM Secretion From CAECs
CAECs spontaneously secreted ADM into the culture medium. Figure 4 illustrates the time course of ADM
secretion in the presence or absence of sarafotoxin S6c
(10-7 mol/L) into the culture medium. ADM
concentration in culture medium from the CAECs increased in a
time-dependent manner up to 24 hours. The spontaneous secretion rate
was 15.7±1.5 pg/105 cells per 24 hours. The
ETB receptor stimulation by sarafotoxin S6c
significantly increased ADM secretion (21.2±1.5
pg/105 cells per 24 hours, P<0.05
versus control secretion). Figure 5
illustrates the dose-response effect of sarafotoxin S6c on the
secretion of ADM. Incubation of the CAECs with sarafotoxin S6c
(10-8 to 10-6 mol/L) for
16 hours increased ADM secretion from the CAECs. Figure 6 illustrates the effects of the
ETA receptor antagonist FR-139317
(10-6 mol/L) and ETB
receptor antagonist IRL-2500 (10-6
mol/L) on the secretion of ADM in the presence and absence of
sarafotoxin S6c (10-9 to
10-7 mol/L) for 16 hours. The
ETB but not ETA receptor
antagonist blocked stimulated secretion of ADM by
sarafotoxin S6c. IRL-2500 alone decreased ADM secretion, suggesting
that basal ET-1 secretion regulates ADM secretion via the
ETB receptor. The decreased ADM secretion by
IRL-2500 was reversed by adding excess sarafotoxin S6c
(10-7 mol/L). Viability of CAECs after 24 hours
of incubation was >98% as determined by trypan blue staining.
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indicates spontaneous secretion of ADM from CAECs; 
, ADM
secretion from CAECs stimulated with sarafotoxin S6c (10-7
mol/L). *P<0.05 between groups.
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P<0.05 vs
S6c.
ET-1 Secretion From CAECs
To determine endogenous ET-1 secretion, which could
stimulate ADM secretion, we measured ET-1 in the culture medium. CAECs
spontaneously secreted ET-1 into the culture medium. Figure 7 (top) illustrates the time course of
ET-1 secretion into the culture medium. ET-1 concentration in culture
medium from the CAECs increased in a time-dependent manner up to 24
hours. The spontaneous secretion rate of ET-1 was 188.3±31.1
pg/105 cells per 24 hours, which is 13 times
higher than ADM secretion rate. Figure 7 (lower panel) illustrates the
relation between ADM and ET-1 concentration in the culture medium,
which demonstrated a positive correlation.
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Northern Blot Analysis
Northern blot analysis of ADM mRNA from the cultured CAECs
in the presence and absence of sarafotoxin S6c is illustrated in Figure 8. Northern blot analysis
demonstrated the presence of ADM mRNA in the cultured CAECs that was
1.6 kb in size, indicating the production of ADM in the
CAECs. The ETB receptor stimulation with
sarafotoxin S6c (10-7 mol/L) significantly
increased ADM gene expression in the cultured CAECs (Figure 8).
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Discussion |
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The objective of the present investigation was to confirm the endothelial cell as a site of presence, production, and secretion of ADM and to define the role of the ETB receptor in the synthesis and release of this peptide. The present findings establish the presence of ADM immunoreactivity in the endothelial cells by immunohistochemistry, ADM production in the endothelial cells by Northern blot analysis, and ADM secretion from the endothelial cells by radioimmunoassay. We have also demonstrated that ADM production and secretion are increased by activation of the ETB receptor in endothelial cells. Furthermore, this enhanced secretory response was blocked by an ETB receptor antagonist. Thus, the present investigation supports an important autocrine interaction between the ADM and ET systems in the control of ADM secretion.
ADM has emerged as a vasoactive peptide originally isolated from pheochromocytoma cells.1 Its biological properties include vasodilatation and natriuresis, with conflicting data related to growth regulation18 19 and inotropism.20 21 While its physiological role continues to emerge, studies suggest its activation in states of cardiorenal dysfunction such as hypertension6 and congestive heart failure,7 8 where it may play a compensatory role to limit excessive vasoconstriction. The present investigation confirms the important concept that ADM is an endothelial cell–derived vasoactive peptide in addition to being secreted by vascular smooth muscle cells. Specifically, the present study, using both immunohistochemical staining of CAECs and radioimmunoassay of cell culture media, establishes the presence and secretion of ADM from the endothelial cell. Thus, ADM emerges as another endothelial cell–derived vasodilatory factor complementing NO, PGI2, and C-type natriuretic peptide.
Sugo et al14 have reported factors that augment ADM secretion from cultured vascular smooth muscle cells. One such factor is ET-1. The present study importantly extends our understanding of an interaction between ET-1 and ADM by demonstrating that stimulation of the ETB receptor with sarafotoxin S6c increases the production and secretion of ADM from the endothelial cells. The ability of an ETB receptor antagonist, IRL-2500, to attenuate the action of sarafotoxin S6c further establishes that ADM secretion is linked to the ETB receptor. Moreover, ETA receptor antagonism with FR-139317 failed to attenuate the action of the ETB agonist. Of interest is the additional observation that IRL-2500 alone significantly decreased ADM secretion, suggesting that via an autocrine action, basal secretion of ET-1 from the endothelial cells regulates ADM secretion via the ETB receptor.
The endocrine role of the ETB receptor has been intensively investigated and indeed may represent the primary role in mediation of the actions of ET-1 under physiological conditions. To date, studies support the ability of the ETB receptor to mediate endothelial release of vasodilatory factors that include NO and PGI2.12 13 The present study extends this endocrine role to ADM. Indeed, these observations may provide some understanding of the continuing vasorelaxing action of ETB stimulation in isolated vessels with endothelium intact when the NO and PGI2 systems are inhibited. Furthermore, it is tempting to speculate that if the endothelium is exposed to physiological stimuli that release ET-1 at very low concentrations, the primary action of the ET system may be in the autocrine release of vasodilatory factors, underscoring a primary vasodilatory rather than constrictor role for ET-1.
Sugo et al5 14 have reported that cultured vascular smooth muscle cells secrete a considerable amount of ADM into the culture medium. Although we did not demonstrate ETB-mediated ADM secretion from the canine vascular smooth muscle cells, ADM is thought to be secreted via ETB receptor, which is also localized to vascular smooth muscle cells.
Recently, cDNA clones encoding human,22 porcine,17 mouse,23 and rat ADM precursor24 were isolated and sequenced. These reports indicate that the structure of mammalian ADM is well conserved among species, and they raise the possibility that antiserum to human ADM could also detect ADM of other mammalian species, such as dogs. In the present study, we used polyclonal anti-human ADM antiserum to detect canine ADM in the CAECs. Although canine ADM has not been isolated, the positive immunostaining substances of the present study are considered to be "canine ADM." With this polyclonal antibody, the present study demonstrates the presence of ADM in CAECs, which may be consistent with the endothelial cells being important sites for ADM production. Northern blot analysis supported this ADM production in CAECs. As for the ETB receptor, although the canine ETB receptor has not been determined, the positive staining for ETB receptor is considered to be "canine ETB receptor" as explained similarly above.
The present study in cultured CAECs may have pathophysiological relevance. To date, in studies of congestive heart failure in which circulating ET-1 is increased, ADM has also been reported to be increased.7 8 It is tempting to speculate that in a state of excessive ET-1 release, as in congestive heart failure, ETB receptor stimulation may explain in part the increase in circulating and tissue ADM7 8 25 reported in such disease states.
In summary, the present study advances our understanding of the vascular biology of ADM, specifically as an endothelial cell–derived vasodilatory peptide. In addition, its secretion from vascular endothelial cells is enhanced by the ETB receptor activation and decreased with ETB receptor blockade. Thus, these studies are the first to establish an autocrine role for the ETB receptor in the release of the vasodilating peptide ADM and suggest another important vasoactive system regulated by the ETB receptor.
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Acknowledgments |
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This work was supported by grants from the American Heart Association, Minnesota Affiliate (MN-97-GB-06), the National Institute of Health (HL 36634), National Kidney Foundation of the Upper Midwest, the Miami Heart Research Institute, and the Bruce and Ruth Rappaport Program in Vascular Biology and Mayo Foundation. We thank Eric E. Williamson, Denise M. Heublein, and Sharon M. Sandberg for excellent assistance. We also acknowledge Todd E. Rasmussen and John B. Osborn for data analysis.
Received June 24, 1998; first decision July 15, 1998; accepted July 22, 1998.
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 T. Tsuruda, M. Jougasaki, G. Boerrigter, B. K. Huntley, H. H. Chen, A. B. D'Assoro, S. C. Lee, A. M. Larsen, A. Cataliotti, and J. C. Burnett Jr Cardiotrophin-1 Stimulation of Cardiac Fibroblast Growth: Roles for Glycoprotein 130/Leukemia Inhibitory Factor Receptor and the Endothelin Type A Receptor Circ. Res., February 8, 2002; 90(2): 128 - 134. [Abstract] [Full Text] [PDF]
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 P. D. Upton, J. Wharton, N. Davie, M. A. Ghatei, D. M. Smith, and N. W. Morrell Differential Adrenomedullin Release and Endothelin Receptor Expression in Distinct Subpopulations of Human Airway Smooth-Muscle Cells Am. J. Respir. Cell Mol. Biol., September 1, 2001; 25(3): 316 - 325. [Abstract] [Full Text] [PDF]
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 P. Kinnunen, J. Piuhola, H. Ruskoaho, and I. Szokodi AM reverses pressor response to ET-1 independently of NO in rat coronary circulation Am J Physiol Heart Circ Physiol, September 1, 2001; 281(3): H1178 - H1183. [Abstract] [Full Text] [PDF]
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 C. B. Taner, S. R. Severson, P. J. M. Best, A. Lerman, and V. M. Miller Treatment with endothelin-receptor antagonists increases NOS activity in hypercholesterolemia J Appl Physiol, March 1, 2001; 90(3): 816 - 820. [Abstract] [Full Text] [PDF]
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 M. Jougasaki and J. C Burnett Jr. Continuing insights into the heart as an endocrine organ: adrenomedullin and cardiac fibroblasts Cardiovasc Res, March 1, 2001; 49(4): 695 - 696. [Full Text] [PDF]
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 T. F. Luscher and M. Barton Endothelins and Endothelin Receptor Antagonists : Therapeutic Considerations for a Novel Class of Cardiovascular Drugs Circulation, November 7, 2000; 102(19): 2434 - 2440. [Abstract] [Full Text] [PDF]
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E. Oie, L. E. Vinge, A. Yndestad, C. Sandberg, H. K. Grogaard, and H. Attramadal Induction of a Myocardial Adrenomedullin Signaling System During Ischemic Heart Failure in Rats Circulation, February 1, 2000; 101(4): 415 - 422. [Abstract] [Full Text] [PDF]
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M. Jougasaki, I. Tachibana, A. Luchner, H. Leskinen, M. M. Redfield, and J. C. Burnett Jr Augmented Cardiac Cardiotrophin-1 in Experimental Congestive Heart Failure Circulation, January 4, 2000; 101(1): 14 - 17. [Abstract] [Full Text] [PDF]
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 G. G. Nussdorfer, G. P. Rossi, L. K. Malendowicz, and G. Mazzocchi Autocrine-Paracrine Endothelin System in the Physiology and Pathology of Steroid-Secreting Tissues Pharmacol. Rev., September 1, 1999; 51(3): 403 - 438. [Abstract] [Full Text] [PDF]
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 E. BROCHU, S. LACASSE-M, R. LARIVIÈRE, I. KINGMA, J. H. GROSE, and M. LEBEL Differential Effects of Endothelin-1 Antagonists on Erythropoietin-Induced Hypertension in Renal Failure J. Am. Soc. Nephrol., July 1, 1999; 10(7): 1440 - 1446. [Abstract] [Full Text]
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T. Tsuruda, M. Jougasaki, G. Boerrigter, B. K. Huntley, H. H. Chen, A. B. D'Assoro, S. C. Lee, A. M. Larsen, A. Cataliotti, and J. C. Burnett Jr Cardiotrophin-1 Stimulation of Cardiac Fibroblast Growth: Roles for Glycoprotein 130/Leukemia Inhibitory Factor Receptor and the Endothelin Type A Receptor Circ. Res., February 8, 2002; 90(2): 128 - 134. [Abstract] [Full Text] [PDF]
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