0022-3565/00/2952-0484$03.00/0
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
JPET 295:484–491, 2000
Vol. 295, No. 2
2926/861060
Printed in U.S.A.
Enhanced EndothelinA Receptor-Mediated Calcium
Mobilization and Contraction in Organ Cultured Porcine
Coronary Arteries1
BRENT J. F. HILL, LAXMANSA C. KATWA, BRIAN R. WAMHOFF, and MICHAEL STUREK
Vascular Biology Laboratory, Dalton Cardiovascular Research Center, and Department of Physiology, School of Medicine, University of
Missouri, Columbia, Missouri (B.J.F.H., B.R.W., M.S.); and Department of Physiology, School of Medicine, East Carolina University, Greenville,
North Carolina (L.C.K.)
Accepted for publication July 31, 2000
This paper is available online at http://www.jpet.org
Endothelin-1 (ET-1) is a potent vasoconstrictor and mitogen of vascular smooth muscle cells (Harrison et al., 1992;
Mathew et al., 1996). Smooth muscle cells contain both the
ETA and ETB receptor subtypes; however, the relative subtype distribution varies depending on the vessel type (Godfraind, 1993; Adner et al., 1998a). In coronary arteries the
ETA receptor subtype predominates; approximately 80% of
the contractile response is attributed to the ETA receptor,
whereas ETB is responsible for the remaining 20% (Dagassan
et al., 1996; Elmoselhi and Grover, 1997).
The selective up-regulation of either subtype is enhanced
with the progression of atherosclerosis, thrombosis, and cardiac hypertrophy (Wang et al., 1995; Mathew et al., 1996;
Stewart, 1998). Evidence suggests either ET receptor subtype may contribute to the enhanced contractile response to
Received for publication May 22, 2000.
1
This study was supported by National Institutes of Health Grants
RR13223 and HL62522 (to M.S.) and an American Heart Association Predoctoral Fellowship (to B.J.F.H.).
and BQ123 (ETA-selective antagonist). Compared with cold
storage, organ culture induced a 2-fold increase in tension
development (3 ⫻ 10⫺7 M ET-1) and Cam (3 ⫻ 10⫺8 M ET-1),
which was inhibited with bosentan, thus confirming the enhanced responses to ET-1 were due to ET receptor activation.
BQ123 also inhibited the enhanced contraction and Cam responses to ET-1. In contrast, BQ788 failed to inhibit tension
development and Cam responses to ET-1 in organ culture and
cold storage. Sarafotoxin 6C (ETB agonist) failed to elicit an
increased Cam response in organ culture compared with cold
storage. Our results indicate the increased tension development and Cam responses to ET-1 in organ culture are attributable to ETA receptors, and not ETB receptors.
ET with arterial injury (Harrison et al., 1992; Wang et al.,
1995; Dagassan et al., 1996; White et al., 1998). Coronary
artery disease is a leading cause of death in the U.S. population (Russell et al., 1998); therefore, it is important to
understand the role of ET-1 receptors in this artery. Few
investigators have studied the functional response to ET-1 in
a coronary artery injury model. Dagassan et al. (1996) described an up-regulation of ETB receptors in the left anterior
descending (LAD) coronary artery from humans displaying
atherosclerosis. Hasdai et al. (1997) also found enhanced
ETB-mediated vasoconstriction in the left circumflex coronary artery from hyperlipidemic pigs. In contrast, Katwa et
al. (1999) and Wang et al. (1995) demonstrated increased
ETA function in the right coronary artery and the LAD coronary artery, respectively, with arterial injury. However,
Godfraind (1993) found that there is a high degree of heterogeneity of ETA and ETB receptors along the length of the
LAD. ET-1-induced contractions are mediated almost entirely by ETA receptors at the distal end of the LAD, whereas
ABBREVIATIONS: ET-1, endothelin-1; ETA, endothelinA; ETB, endothelinB; LAD, left anterior descending coronary artery; Cam, myoplasmic
calcium; DAPI, 4⬘,6-diamidino-2-phenylindole dihydrochloride; PSS, physiological salt solution; 80K, 80 ⫻ 10⫺3 M KCl solution; Tmax, response
to a maximal concentration of an agonist; SR, sarcoplasmic reticulum; OC, organ culture; CS, cold storage.
484
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ABSTRACT
Arterial injury models for coronary artery disease have demonstrated an enhanced expression and function of either the
endothelinA or endothelinB (ETA or ETB) receptor subtype. We
hypothesized that organ culture would enhance the physiological function of ET receptors in the porcine right coronary artery.
Arteries were either cold stored (4°C) or organ cultured (37°C)
for 4 days. After 4 days, the artery was either 1) sectioned into
rings to measure the ET-1-induced isometric tension response
(3 ⫻ 10⫺10–3 ⫻ 10⫺7 M), or 2) enzymatically dispersed and the
isolated smooth muscle cells imaged using fura-2 to measure
the myoplasmic calcium (Cam) response to 3 ⫻ 10⫺8 M ET-1
(⬃EC50). Isometric tension and Cam to ET-1 were measured in
the absence and presence of bosentan (nonselective ETA or
ETB receptor antagonist), BQ788 (ETB-selective antagonist),
2000
Enhanced ETA Receptor Function with Organ Culture
Materials and Methods
Organ Culture and Single-Cell Fura-2 Digital Imaging.
Hearts were obtained from local abattoirs and prepared as described
in our laboratory (Sturek et al., 1991). Right coronary arteries (distal
end) were either cold stored for 4 days at 5°C or organ cultured for 4
days at 37°C in a 95% O2, 5% CO2 incubator. The arterial segments
that were organ cultured or cold stored were opened longitudinally to
expose the lumen and placed with the lumen facing up in a 100-mm
Petri dish containing 30 ml of RPMI 1640 (Life Technologies, Grand
Island, NY). The RPMI 1640 was changed every 2 days.
Smooth muscle cells from cold-stored or organ-cultured arteries
were isolated in a vial containing an enzymatic solution, which was
replaced every 60 min, with the following composition (Sturek et al.,
1991): 294 U/ml collagenase (Worthington, Freehold, NJ), 2 mg/ml
bovine serum albumin (Sigma Chemical Co., St. Louis, MO), 1 mg/ml
soybean trypsin inhibitor (Worthington), and 0.4 mg/ml DNase I
(type IV; Sigma Chemical Co.). After 2 or 3 h the dispersed cells in
the collagenase solution primarily contained smooth muscle cells,
and were identified morphologically (Wagner-Mann et al., 1992).
Cam levels were measured using the InCa⫹⫹ calcium imaging
system (Intracellular Imaging Inc., Cincinnati, OH), and similar to
previously published methods (Sturek et al., 1991, 1992). Briefly, the
cells were incubated with 2.5 ⫻ 10⫺6 M fura-2-acetoxymethyl ester
at 37°C for 25 min. In those experiments using antagonists, cells
were incubated for 1 h with the appropriate concentration of the
antagonist before commencing with single-cell digital imaging. A
drop of the fura-2-loaded cellular suspension was placed on a coverslip inside a constant flow superfusion chamber (Science Instruments Shop, University of Missouri, Columbia, MO), which was
mounted on an inverted epifluorescence microscope (model TMS-F;
Nikon, Melville, NY). Cells were allowed to settle and adhere to the
coverslip (Fig. 5A, inset) before commencing with the experiments.
Fura-2 was excited by 340- and 380-nm light and the emitted fluorescence (510 nm) collected by a monochrome charge-coupled device
camera (Cohu, Inc., San Diego, CA) that was attached to a computer
for data aquisition by the InCa ratiometric fluorescence program,
version 1.2 (Intracellular Imaging Inc.). Data are expressed as a
ratio (and indicated as ratio units) of the emitted light intensity at
340- and 380-nm excitation rather than [Ca2⫹m] because of uncertainties, mainly impaired calcium sensitivity, detailed in previous
reports (Sturek et al., 1991, 1992; Wagner-Mann et al., 1991, 1992).
An especially important consideration is that the “in situ” calibration
is similar for cells from cold-stored and organ-cultured arteries (data
not shown); thus, fura-2 fluorescence ratios are directly comparable
between groups whether calcium sensitivity is altered or not. The in
situ calibration provides a better representation concerning the state
of the acetoxymethyl ester form of fura-2 compared with the free acid
form of fura-2 in cells in a simplified mock intracellular solution.
DNA Imaging. Cold-stored and organ-cultured arteries were
fixed in 4% paraformaldehyde and incubated for 24 h in 30% sucrose
at (20°C). Arteries were freeze mounted and cut into 7-␮m sections.
Cross sections were stained with 2.5 ⫻ 10⫺7 M 4⬘,6-diamidino-2phenylindole dihydrochloride (DAPI) for 20 min at 37°C to quantitate single-cell DNA content (Kapuscinski, 1995). Imaging of DAPI
fluorescence was done using a widefield epifluorescent microscope
(Nikon Diaphot, Garden City, NY). Images at three focal planes 0.5
␮m apart in the z-axis were acquired for deconvolution analysis.
Out-of-focus fluorescence was removed by deconvolution software
equipped with a digital signal processing board that used the nearest
neighbor algorithm (Vaytek, Inc., Fairfield, IA). DAPI fluorescence
was quantitated using Image Pro Plus 3.0 software (Media Cybernetics, Silver Springs, MD).
Isometric Tension Measurements. Similar to the single-cell
imaging experiments, porcine right coronary arteries were sectioned
and placed in a Petri dish containing RPMI 1640 for cold storage or
organ culture. After 4 days, vessel segments were sectioned into
5-mm rings and the endothelium removed using a toothpick. Rings
were also further studied within 3 h after the sacrifice of the pig. The
absence of an intact endothelium was confirmed by the lack of a
relaxation response to 1 ⫻ 10⫺7 M bradykinin. Rings were mounted
via two stainless steel wire supports in 25 ml of isolated organ baths
maintained at 37°C, and the physiological salt solution (PSS) aerated
with a 95% O2, 5% CO2 mixture. The two support wires were connected to an isometric force transducer (Grass Medical Instruments,
Quincy, MA) and a linear displacement micrometer (Mitutoyo, MTI
Corp., Paramus, NJ). Force generation was amplified by a customdesigned amplifier (Technical Resources Core Facility, Dalton Cardiovascular Research Center), and the data acquired by a computer
equipped with an analog-to-digital converter and Labtech Acquire
software (Laboratory Technologies Corp., Wilmington, MA). Rings
were set near their optimum length-tension relationship by progressively lengthening each ring and subsequently contracting it with
the addition of a 60 ⫻ 10⫺3 M KCl solution to the organ bath. This
procedure was repeated until the active force generated was no more
than 10% greater than at the previous length. Rather than stretching all vessel rings to the same passive tension, the method of setting
each vessel at optimal length was chosen to reduce variability and
maximize the response of each vessel ring. Rings were initially
exposed to an 80 ⫻ 10⫺3 M KCl solution (80K) before exposing them
to ET-1. In those experiments using antagonists, cells were incubated for 45 min with the appropriate concentration of the antagonist before generating a cumulative concentration-response relationship to ET-1 in half-log increments (3 ⫻ 10⫺10–3 ⫻ 10⫺7 M).
To normalize each individual arterial response to ET-1, the ET-1induced tension development is expressed as a percentage of the
maximum contractile response generated by 80K. Overall, there was
no difference (P ⬎ .05) in the absolute tension (grams) developed to
80K in cold-stored and organ-cultured arterial rings.
Solutions. Isolated smooth muscle cells within the superfusion
chamber were continually superfused with PSS containing 138 ⫻
10⫺3 M NaCl, 5 ⫻ 10⫺3 M KCl, 2 ⫻ 10⫺3 M CaCl2, 1 ⫻ 10⫺3 M MgCl2,
10 ⫻ 10⫺3 M HEPES, 10 ⫻ 10⫺3 M glucose, titrated to pH 7.4 (with
NaOH). The depolarizing solution (80K or 25 ⫻ 10⫺3 M KCl solution)
was composed of 65 ⫻ 10⫺3 M NaCl, 80 or 25 ⫻ 10⫺3 M KCl, 2 ⫻ 10⫺3
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ETB receptors are partially responsible for the contraction at
the proximal end. To eliminate this large degree of heterogeneity we used the right coronary artery because it has a
predominant ETA receptor population that is responsible for
mediating vasoconstriction (Bacon and Davenport, 1996;
Schiffrin and Touyz, 1998). In this study we hypothesized
that arterial injury (i.e., organ culture) would enhance ET
receptor-mediated vasoconstriction and myoplasmic calcium
(Cam) responses in the porcine right coronary artery.
Organ culture is a technique that has been used to study
cell proliferation in vessels (Gotlieb and Boden, 1984; Newby
and Zaltsman, 1999; Voisard et al., 1999). Newby and Zaltsman (1999) recently demonstrated medial proliferation of
smooth muscle cells within the organ-cultured rabbit and pig
aorta, as well as human saphenous veins. They indicate that
organ culture greatly parallels chemotaxis and matrixes remodeling in animal models because of the intact interactions
that are present within organ culture. Recently, Voisard et
al. (1999) pointed out that organ culture is a valuable model
that mimics the injury response of atherosclerosis.
This study uniquely demonstrates that the Cam response
mediated by ETA or ETB receptors in isolated smooth muscle
cells is paralleled by the development of isometric tension in
both cold-stored and organ-cultured coronary arteries. Our
results indicate that the 2-fold increase in Cam and tension
development to ET-1 with organ culture is attributed to ETA
receptors, and not ETB receptors.
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Hill et al.
Vol. 295
⫺3
⫺3
⫺3
Results
A concentration-response relationship (3 ⫻ 10⫺10–3 ⫻
10⫺7 M) to ET-1 was generated in fresh (n ⫽ 8) and coldstored arterial rings (n ⫽ 18). The pD2 values (⫺log EC50) in
fresh and cold-stored rings (7.96 ⫾ 0.23 and 7.60 ⫾ 0.13,
respectively) were not significantly different. Additionally,
isometric tension development (96 ⫾ 12 and 121 ⫾ 8%, respectively) to a maximal concentration (Tmax) of ET-1 (3 ⫻
10⫺7 M) was not significantly different between fresh and
cold-stored rings. We have previously demonstrated (M.
Sturek, B. Hill, and B. Wamhoff, unpublished observations)
using isolated single smooth muscle cells from fresh and
cold-stored arteries that there is no difference in the basal
Cam concentration or the peak Cam response to 5 ⫻ 10⫺3 M
caffeine or 80K between groups, suggesting cold-stored arteries serve as an adequate paired time control for those
arteries that were organ cultured.
A cumulative concentration-response relationship to ET-1
was generated from 3 ⫻ 10⫺10 to 3 ⫻ 10⫺7 M in arterial rings
that had been organ cultured (n ⫽ 20). As demonstrated in
Fig. 1, organ-cultured rings demonstrated a significant increase (227 ⫾ 20%) in their Tmax to 3 ⫻ 10⫺7 M ET-1 compared with cold-stored rings (121 ⫾ 8%; n ⫽ 18). However,
there was no difference in potency (pD2) to ET-1 between
cold-stored and organ-cultured rings (7.60 ⫾ 0.13 and 7.56 ⫾
0.03, respectively). The 2-fold increase in the Tmax of organcultured rings indicates organ culture may enhance the physiological function of ET receptors to elicit a contraction. To
identify whether either the ETA or ETB subtype contributes
to this increased tension development, a concentration-response relationship was generated to ET-1 in the absence
Fig. 1. Organ-cultured arterial rings display an enhanced contractile
response to ET-1 compared with cold-stored rings. ET-1-induced tension
development is expressed as a percentage (%) of the maximum contractile
response generated by 80K. ⴱ, represents a significant (P ⬍ .05) difference
from the cold-stored rings.
and presence of bosentan (ETA and ETB antagonist),
PD145065 (ETA and ETB antagonist), BQ123 (ETA antagonist), and BQ788 (ETB antagonist).
Bosentan (10⫺6 M), a nonselective ET antagonist, inhibited
isometric tension development to ET-1 in organ-cultured and
cold-stored arterial rings as indicated by the significant
rightward shift of the concentration-response relationship to
ET-1 (Fig. 2; Table 1). In cold-stored rings, the pD2 value in
the absence of bosentan was 7.95 ⫾ 0.10. The pD2 could not
be calculated in cold-stored rings in the presence of bosentan
because a sigmoidal concentration-response relationship was
not generated. In organ-cultured rings, the pD2 values in the
absence and presence of bosentan were 7.57 ⫾ 0.05 and
6.78 ⫾ 0.14, respectively. Bosentan did not significantly affect the Tmax in organ-cultured and cold-stored rings. In
addition, another ETA and ETB antagonist, PD145065 (10⫺6
M), similarly shifted the ET-1 concentration-response relationship to ET-1 to the right in both cold-stored and organcultured rings (data not shown).
Fig. 2. Antagonism of contractions to ET-1 by bosentan in organ-cultured
(OC) and cold-stored (CS) arterial rings. A cumulative concentrationresponse relationship was generated to ET-1 (3 ⫻ 10⫺10–3 ⫻ 10⫺7 M) in
the absence or presence of 10⫺6 M bosentan in organ-cultured and coldstored rings. The ET-1-induced tension development is expressed as a
percentage (%) of the maximum contractile response generated by 80K. ⴱ,
indicates a significant (P ⬍ .05) difference in tension development from
OC ⫹ bosentan at each respective ET-1 concentration. ⵩, indicates a
significant difference in tension development from CS ⫹ bosentan.
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M CaCl2, 1 ⫻ 10 M MgCl2, 10 ⫻ 10 M HEPES, 10 ⫻ 10 M
glucose, titrated to pH 7.4 (with NaOH). For isometric tension measurements the PSS was similar to that for single cells except that
24 ⫻ 10⫺3 M NaHCO3 was added and NaCl was reduced by 24 ⫻
10⫺3 M. The stock solution of potassium chloride (Sigma Chemical
Co.) was prepared in distilled water. Fura-2-acetoxymethyl ester
(Molecular Probes, Inc., Eugene, OR), BQ123 (Peptides International, Louisville, KY), PD145065 (Sigma Chemical Co.), and BQ788
(Peptides International) were prepared in dimethyl sulfoxide (Sigma
Chemical Co.). DAPI (Molecular Probes, Inc.) and endothelin-1 (Peninsula Labs, Inc., San Carlos, CA) were prepared in N,N-dimethylformamide (Sigma Chemical Co.) and 0.01 N acetic acid, respectively. Bosentan was a gift from Roche Pharmaceuticals (Nutley,
NJ). Drugs to be used within the superfusion system were diluted
from stock solutions into PSS and superfused at a rate of approximately 2 ml/min.
Statistical Analysis. Data were expressed as the mean ⫾ S.E. for
the number (n) of single cells (fura-2 imaging) or animals (isometric
tension) within each group. Analysis of data was done by either a
one-way ANOVA or a Kruskal-Wallis one-way ANOVA followed by
Bonferroni’s test or Dunn’s test, respectively, when more than two
groups were present. A paired or unpaired Student’s t test was used
when comparing only two groups. Smooth muscle cells that were
identified as “responders” to an agonist were defined as those cells
whose response to an agonist was at least three standard deviations
above the baseline for 5% of the time exposed to the agonist (P ⬍ .01).
The percentage of responders was analyzed using the chi square
distribution. Statistical analyses of the data were performed using
SigmaStat (Jandel Scientific Software, San Rafael, CA). The pD2
values (⫺log EC50) were calculated and analyzed using GraphPad
Prism 2.0 (GraphPad Software Inc., San Diego, CA). Significance
was defined as P ⬍ .05.
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Enhanced ETA Receptor Function with Organ Culture
487
TABLE 1
Antagonism of ET-1-induced tension development
Condition
Antagonist
n
pD2a
Tmaxb
Cold stored
Control
Bosentan
Control
BQ123 (10⫺6
Control
BQ123 (10⫺5
Control
BQ788
Control
Bosentan
Control
BQ123 (10⫺6
Control
BQ123 (10⫺5
Control
BQ788
5
5
5
5
4
4
3
3
6
6
5
5
5
5
3
3
7.95 ⫾ 0.10
—c
7.48 ⫾ 0.09
7.39 ⫾ 0.09
7.53 ⫾ 0.11
—
7.67 ⫾ 0.14
7.71 ⫾ 0.18
7.57 ⫾ 0.05
6.78 ⫾ 0.14d
7.60 ⫾ 0.05
6.92 ⫾ 0.06d
7.67 ⫾ 0.04
6.81 ⫾ 0.12d
7.36 ⫾ 0.11
7.53 ⫾ 0.15
103 ⫾ 25
77 ⫾ 42
132 ⫾ 5
108 ⫾ 18
123 ⫾ 11
32 ⫾ 10d
134 ⫾ 14
139 ⫾ 18
519 ⫾ 148
284 ⫾ 139
197 ⫾ 19
206 ⫾ 17
215 ⫾ 66
148 ⫾ 51
314 ⫾ 59
288 ⫾ 16
Organ culture
M)
M)
M)
M)
Each value is the pD2 ⫾ S.E. that represents the ⫺log EC50 for n animals.
b
Each value is the Tmax ⫾ S.E. that indicates the response to a maximal concentration of ET-1 (3 ⫻ 10⫺7 M) for n animals. The data are expressed as a percentage of
the maximum contractile response to 80 mM KCl.
c
—, a sigmoidal concentration-response relationship was not generated, so the EC50 could not be calculated.
d
P ⬍ .05 from the paired control preparation.
a
Fig. 3. Antagonism of contractions to ET-1 by BQ123 in OC and CS
arterial rings. A, inhibition of ET-1-induced tension development by 10⫺5
M BQ123 in organ-cultured and cold-stored rings. B, inhibition of ET-1induced tension development by 10⫺6 M BQ123 in organ-cultured rings.
A cumulative concentration-response relationship was generated to ET-1
(3 ⫻ 10⫺10–3 ⫻ 10⫺7 M) in the absence or presence of either 10⫺5 or 10⫺6
M BQ123 in organ-cultured and cold-stored rings. The ET-1-induced
tension development is expressed as a percentage (%) of the maximum
contractile response generated by 80K. ⴱ, indicates a significant (P ⬍ .05)
difference in tension development from OC ⫹ BQ123 at each respective
ET-1 concentration. ⵩, indicates a significant difference in tension development from CS ⫹ BQ123.
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Selective inhibition of isometric tension development was
determined using the selective ETA antagonist BQ123 (Table
1). In cold-stored rings, only 10⫺5 M BQ123 (Fig. 3A) significantly inhibited the concentration-response relationship to
ET-1; no inhibition was demonstrated using 10⫺6 M BQ123
(Fig. 3B). In contrast, both 10⫺6 M (Fig. 3B) and 10⫺5 M (Fig.
3A) BQ123 shifted the concentration-response relationship
(i.e., pD2 values) to the right 5- and 7-fold, respectively, in
organ-cultured rings. As shown by Fig. 4, selective inhibition
of the ETB receptor with BQ788 (10⫺5 or 10⫺6 M BQ788) did
not alter the pD2 value or the Tmax in the cold-stored and
organ-cultured arterial rings (Table 1). Using cardiac fibroblasts we have confirmed the antagonistic activity of BQ788,
which contain both ETA and ETB receptors (data not shown).
To further investigate the altered contractile function of
ET receptors, smooth muscle cells were enzymatically isolated from both cold-stored and organ-cultured arteries, and
the Cam response measured to 3 ⫻ 10⫺8 M ET-1 in the
absence and presence of 10⫺5 M bosentan, BQ123, and
BQ788 (Fig. 5, A and B). We used 3 ⫻ 10⫺8 M ET-1 because
this was the approximate EC50 value (pD2 ⫽ 7.5) for the
concentration-response relationship generated to ET-1 in
cold-stored and organ-cultured arterial rings. Previous observations indicate that not all cells respond to ET-1 in the
absence of an antagonist; however, in evaluating the effect of
antagonists on the Cam response all cells (both responders
and nonresponders) were pooled. All cells were pooled because it would not be known whether the nonresponders to
ET-1 inherently did not respond to ET-1, or whether the
antagonist effectively inhibited the Cam response to ET-1.
Because our reported results include both responders and
nonresponders to ET-1, the reported Cam response to ET-1
will be lower (due to the “dilution” of the Cam response to
ET-1 by the nonresponders) than if calculated for just those
cells responding to ET-1. Similar to the development of isometric tension in arterial rings, cells from organ-cultured
arteries had a significantly increased Cam response to ET-1
(0.50 ⫾ 0.05 ratio units; n ⫽ 20) compared with those cells
from cold-stored arteries (0.19 ⫾ 0.03 ratio units; n ⫽ 24).
Bosentan significantly decreased the ET-1-induced Cam response in cells from both cold-stored (0.09 ⫾ 0.02 ratio units;
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Hill et al.
Vol. 295
Fig. 4. There is no antagonism of ET-1-induced contractions by BQ788 in
OC and CS arterial rings. A cumulative concentration-response relationship was generated to ET-1 (3 ⫻ 10⫺10–3 ⫻ 10⫺7 M) in the absence or
presence of 10⫺ 5 M BQ788 in organ-cultured and cold-stored rings. The
ET-1-induced tension development is expressed as a percentage (%) of the
maximum contractile response generated by the 80K.
Fig. 5. Inhibition of ET-1-induced increases in myoplasmic calcium by
several selective ET-1 antagonists. A, representative tracing from a single smooth muscle cell isolated from an organ-cultured artery and exposed to 3 ⫻ 10⫺8 M ET-1. An example of smooth muscle cells to be
digitally imaged is demonstrated in the inset. The black line surrounding
a single cell represents the enclosed “area of interest” (i.e., cell area) to be
measured and analyzed. B, antagonism of ET-1-induced increases in
myoplasmic calcium in isolated smooth muscle cells from cold-stored and
organ-cultured arteries. In those experiments using antagonists, cells
were incubated for 1 h with 10⫺5 M of the antagonist before exposing
them to 3 ⫻ 10⫺8 M ET-1. ⴱ, indicates a significant (P ⬍ .05) difference
from the no antagonist condition within the organ-cultured or cold-stored
group. ⵩, indicates a significant difference from the cold-stored, noantagonist group.
influx (which has a negligible contribution to the peak Cam
response) via voltage-gated calcium channels after the initial
release of the SR calcium store. In our study, the minimal
contribution of calcium influx to the peak Cam response is
demonstrated by the absence of a sustained “plateau” phase
after the initial transient calcium spike (due to SR calcium
release; Fig. 5A) after ET-1 application in cells from both
cold-stored and organ-cultured arteries. In our preparation,
this lack of a measurable sustained calcium influx in response to ET-1 is due to the rapid extrusion of calcium from
the cell (Rasmussen et al., 1989; Bowles et al., 1995). We
demonstrated that ET-1-induced tension development is, in a
large part, due to sustained calcium influx by exposing arte-
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n ⫽ 29) and organ-cultured (0.10 ⫾ 0.03 ratio units; n ⫽ 14)
arteries compared with the paired control cells without
bosentan (0.19 ⫾ 0.03 and 0.50 ⫾ 0.05 ratio units, respectively). In cells from organ culture, BQ123 also significantly
decreased the Cam response (0.13 ⫾ 0.04 ratio units; n ⫽ 14)
to similar levels as bosentan. BQ788 had little effect (P ⬎ .05)
on the ET-1 response (0.37 ⫾ 0.05 ratio units; n ⫽ 18) in cells
from organ culture. In cells from cold-stored arteries, both
BQ123 (0.19 ⫾ 0.04 ratio units; n ⫽ 19) and BQ788 (0.27 ⫾
0.03 ratio units; n ⫽ 20) did not significantly decrease the
Cam response to ET-1.
In agreement with data generated by both isometric tension recordings and single-cell Cam responses to ET-1 in the
presence of BQ788, the selective ETB receptor agonist sarafotoxin 6C (10⫺8 M), did not significantly increase Cam above
basal levels in isolated cells from cold-stored and organcultured arteries (Fig. 6). Sarafotoxin 6C has been extensively used as an ETB agonist in vascular smooth muscle
(Adner et al., 1998a; White et al., 1998). We previously found
that 10⫺8 M sarafotoxin 6C elicited a similar Cam response as
10⫺7 M sarafotoxin 6C. Previously, we determined that not
all cells respond to ET-1 and sarafotoxin 6C; therefore, the
Cam response was determined only in those cells that responded to the agonists (Fig. 6). In cells isolated from organcultured arteries, 75% (15 of 20) responded to ET-1, whereas
only 14% (5 of 35) responded to sarafotoxin 6C. However, in
cells from cold-stored arteries, 33% (4 of 12) and 35% (9 of 26)
of the cells responded to ET-1 and sarafotoxin 6C, respectively. As shown in Fig. 6, ET-1 elicited a significantly increased Cam response of 0.47 ⫾ 0.10 ratio units above baseline in isolated cells from organ culture compared with the
Cam response in cells from cold-stored arteries (0.07 ⫾ 0.03
ratio units). In contrast to ET-1, the Cam response to sarafotoxin 6C was significantly decreased with organ culture
(0.04 ⫾ 0.03 ratio units) compared with cold storage (0.11 ⫾
0.04 ratio units).
This study, as well as previous studies by our lab (WagnerMann and Sturek, 1991, 1992), indicate that the peak Cam
response elicited to ET-1 in isolated smooth muscle cells was
predominantly due to the release of calcium from the sarcoplasmic reticulum (SR). However, our lab and others (Goto et
al., 1989; Wagner-Mann and Sturek, 1991) have shown that
ET-1 does appear to induce a very small amount of calcium
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Enhanced ETA Receptor Function with Organ Culture
489
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Fig. 6. Organ culture enhances and attenuates the myoplasmic calcium
responses to ET-1 and sarafotoxin 6C, respectively, compared with cold
storage. Isolated smooth muscle cells from organ-cultured or cold-stored
arteries were either exposed to 3 ⫻ 10⫺8 M ET-1 or 10⫺8 M sarafotoxin
6C. ⴱ, indicates a significant (P ⬍ .05) difference from the organ culture
group exposed to ET-1. ⵩, indicates a significant difference from the
organ culture groups exposed to sarafotoxin 6C.
rial rings to ryanodine (10⫺5 M) for 50 min, which our lab
previously documented mobilizes and fully depletes the SR
calcium store (Wagner-Mann and Sturek, 1991, 1992). Subsequent to ryanodine, 5 ⫻ 10⫺8 M ET-1 was applied for 25
min, which induced a significant contractile response.
Cold-stored (Fig. 7A; n ⫽ 5) and organ-cultured (Fig. 7B;
n ⫽ 5) artery sections were stained with DAPI, an indicator
of DNA content. DAPI fluorescence has extensively been
used to measure nuclear DNA content in cells (Seiler et al.,
1993; Kapuscinski, 1995). Previous investigators have confirmed that the relative DAPI fluorescence intensity is directly related to both DNA content (Seiler et al., 1993) and
cell proliferation (McCaffrey et al., 1988). There was a significant increase in the amount of DAPI fluorescence in single smooth muscle cells (Fig. 7C) from the cross-sectioned,
organ-cultured (164.60 ⫾ 3.55) compared with cold-stored
(96.09 ⫾ 2.64) arteries. In addition, DAPI-stained nuclei in
organ-cultured cross sections (Fig. 7B) demonstrated a lack
of circumferential orientation compared with the circumferentially oriented cold-stored cells (Fig. 7A).
Discussion
Previous investigators have demonstrated an enhanced
expression and function of either the ETA or ETB receptor
subtype in different models of arterial injury (Wang et al.,
1995; Dagassan et al., 1996; Mathew et al., 1996; Hasdai et
al., 1997; Stewart, 1998; Katwa et al., 1999). We hypothesized that there is an enhanced physiological function of ET
receptors in an organ culture model of the porcine right
coronary artery. Although no studies have directly addressed
the possibility that smooth muscle cells may respond differently to ET-1 if they are isolated from the artery itself, this
study uniquely demonstrates that isolated single smooth
muscle cells respond similarly as those smooth muscle cells
possessing cell-cell interactions within the vessel wall. Our
results demonstrate that both intact arterial rings and isolated smooth muscle cells display an enhanced response (i.e.,
tension development and Cam response, respectively) to ET-1
Fig. 7. Arterial cross sections from organ-cultured arteries exhibit increased DNA content. Cold-stored and organ-cultured arteries were cut
into 7-␮m sections and incubated with 2.5 ⫻ 10⫺7 M DAPI (DNA stain).
A, typical cold-stored artery section stained with DAPI. The white arrows
(A and B) indicate DAPI fluorescence of single smooth muscle cells. Cells
from cold-stored arteries run circumferentially as indicated by the elongated cell morphology. B, typical organ-cultured artery section stained
with DAPI. Compared with A, smooth muscle cells in B are disoriented,
which is characteristic of smooth muscle cell migration. C, mean DAPI
fluorescence of single smooth muscle cells within each cross section.
with organ culture. Specifically, this study demonstrates that
organ culture enhances the physiological function of ETA
receptors in the right coronary artery.
The organ culture model has been extensively used to
490
Hill et al.
kinase C (Assender et al., 1996; Schiffrin and Touyz, 1998;
Suzuki et al., 1999). Both Douglas et al. (1994) and McKenna
et al. (1998) have demonstrated that ET-1 promotes neointimal thickening in arterial injury models. Therefore, the enhanced release of calcium from the SR with organ culture
may induce cell proliferation as is demonstrated during atherosclerotic development.
We originally hypothesized that the enhanced tension development and Cam increase in response to ET-1 with organ
culture was due, in part, to an enhanced action of ET receptors. This was confirmed using the nonselective ET antagonist bosentan, which inhibited both tension development in
arterial rings and the Cam response in isolated single cells.
Bosentan shifted the concentration-response relationship to
the right 6-fold and inhibited the Cam response by 80% in
cells isolated from organ-cultured arteries. Therefore, our
results corroborate those reports by investigators who have
shown that ET-1 receptors are up- or down-regulated in
response to a variety of vascular pathologies and organ culture (Wang et al., 1995; Adner et al., 1996, 1998a,b; Mathew
et al., 1996; Stewart, 1998).
We determined the contribution of the ETA receptor subtype in mediating the enhanced contractile and Cam response
in organ-cultured arteries using the selective ETA antagonist
BQ123. In cold-stored rings 10⫺6 M BQ123 was ineffective in
inhibiting the ET-1 response; only 10⫺5 M BQ123 inhibited
the ET-1 response. In contrast, both 10⫺6 and 10⫺5 M BQ123
were effective in shifting the concentration-response relationship 5- and 7-fold to the right, respectively, in organcultured rings. This is similar to the 6-fold shift to the right
with bosentan in organ-cultured rings. Because 10⫺6 M
BQ123 inhibited and had no effect on organ-cultured and
cold-stored rings, respectively, this suggests that the increased contractile response to ET-1 is principally due to ETA
receptors in organ-cultured arteries.
Isolated single cells demonstrated a similar sensitivity to
BQ123 as displayed by organ-cultured arterial rings. Similar
to bosentan, BQ123 decreased the Cam response approximately 80% in cells from organ culture. In cells from coldstored arteries, BQ123 did not decrease the Cam response,
whereas bosentan did decrease Cam by about 50%. In evaluating the effect of the antagonists on the Cam response we
pooled both cells that did or did not respond to ET-1 in the
absence and presence of the antagonist. Otherwise, in the
presence of the antagonist, it would not be known whether
the cells inherently did not respond to ET-1, or whether the
antagonist effectively inhibited the ET-1 response. This dilution of the response to ET-1 may contribute to the apparent
lack of ET-1 inhibition by BQ123 in cells from cold-stored
arteries. Because only 33% of the cells from cold-stored arteries responded to ET-1 (no antagonist) this decreased the
overall reported Cam response, which makes it difficult to
formulate a definitive conclusion regarding the antagonism
of the ET-1 Cam response in these cells. In contrast, because
75% of the cells from organ-cultured arteries responded to
ET-1 (nonantagonist) there was little dilution of the Cam
response to ET-1, therefore the antagonism of ET-1 is more
apparent and definitive.
Unlike BQ123, the selective ETB antagonist BQ788 did not
inhibit isometric tension development or the Cam response to
ET-1 in both intact rings and isolated cells from organ-cultured or cold-stored arteries. In response to the selective ETB
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study cell proliferation and migration (Gotlieb and Boden,
1984; Newby and Zaltsman, 1999; Voisard et al., 1999). We
uniquely confirmed that cells from organ-cultured arteries
are undergoing cell cycling because in intact arterial cross
sections there is a greater DNA content than cells from cold
storage. This agrees with investigators who confirmed that
the relative DAPI fluorescence intensity positively correlates
with cell proliferation (McCaffrey et al., 1988). Furthermore,
the DAPI staining in single cells from organ-cultured arterial
cross sections demonstrated a lack of orientation compared
with the circumferentially oriented cold-stored cells. The
cells from organ-cultured arteries appear to be in a migrating
state. Recently, organ culture has also been used to study
receptor function and density within these proliferating cells,
such as happens in atherosclerosis (Adner et al., 1996,
1998a,b). Therefore, we used the organ culture model to
determine whether the contractile activity of coronary
smooth muscle cells is altered with arterial injury.
Our results indicate that there was no difference in the
potency of ET-1 if the artery was organ cultured. Our pD2
values for cold-stored and organ-cultured rings were 7.60 ⫾
0.13 and 7.56 ⫾ 0.03, respectively (P ⬎ .05). This agrees with
Harrison et al. (1992) who reported a pD2 value of 8.17 by
measuring tension development in endothelium-denuded
rings from pig coronary arteries. Because the pD2 values
were similar between cold-stored and organ-cultured rings, it
might be assumed that a maximal concentration of ET-1 (3 ⫻
10⫺7 M) would elicit a similar contractile response between
groups as was shown by Adner et al. (1995) using fresh and
organ-cultured human omental arteries. However, we demonstrated an approximate 2-fold increase in isometric tension
development to a maximal concentration of ET-1 in organcultured arterial rings compared with cold-stored rings. In
addition, using the ET-1 EC50 (as determined in the arterial
rings) we showed that ET-1 increased the Cam response
2.6-fold in single cells that were isolated from organ-cultured
arteries compared with those isolated from cold-stored arteries. The apparent higher sensitivity to ET-1 in isolated single
smooth muscle cells than in intact arterial rings appears to
be due to the source of calcium used for tension development.
Our data suggest that contractile force is highly dependent
on calcium influx, and not SR calcium release. This suggests
that the greater contractile force generated in organ-cultured
rings is partially attributed to an increase in calcium influx.
Other studies have also shown that ET-1-induced calcium
influx mediates tension development (Kasuya et al., 1989;
Inui et al., 1999). It has been demonstrated that a sustained
influx of calcium is matched by calcium extrusion (so-called
“calcium cycling”), which returns bulk Cam to basal levels
without limiting sustained tension development (Rasmussen
et al., 1989; Bowles et al., 1995). Therefore, because the
ET-1-induced influx of calcium is rapidly extruded from the
cell (Bowles et al., 1995), ET-1-induced calcium influx is not
apparent using single-cell digital imaging of bulk Cam. However, we used single-cell fura-2 digital imaging to measure
SR calcium release (Wagner-Mann and Sturek, 1991, 1992).
Our data suggest that there is a greater enhancement of the
ET-1-mediated release of calcium from the SR with organ
culture. Investigators have found that the transient ET-1
induced intracellular release of calcium (via inositol triphosphate) appears to be primarily linked to mitogenesis and
sensitization of the myofilaments via the activation of protein
Vol. 295
2000
Acknowledgments
We thank Julie Childress and Qicheng Hu for technical assistance.
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agonist sarafotoxin 6C, cells from organ-cultured arteries
actually decreased their Cam response 60% compared with
cells from cold storage. These Cam data suggest there may be
a functional down-regulation of ETB receptors. However, it is
difficult to accurately conclude there is a decreased function
of ETB receptors with organ culture because our data demonstrate such a small Cam response to sarafotoxin 6C and
very little contribution of ETB-mediated tension development
in both normal and organ-cultured arteries.
Our results are important because ET receptors have been
implicated in atherosclerosis (Mathew et al., 1996) and neointimal formation after balloon angioplasty (Douglas et al.,
1994). An oral ETA antagonist reduced neointimal hyperplasia in a porcine model of coronary artery injury (McKenna et
al., 1998). In addition, Katwa et al. (1999) found that the
expression of ETA receptors is up-regulated in porcine coronary arteries during restenosis. In contrast, Dagassan et al.
(1996) described an up-regulation of ETB receptors in atherosclerotic human coronary arteries. However, Dagassan et
al. (1996) used the left anterior descending coronary artery,
which appears to have a greater percentage of ETB receptors
than the right coronary artery (Harrison et al., 1992; Godfraind, 1993; Elmoselhi and Grover, 1997; Hasdai et al.,
1997). The right coronary artery, as was used in this study,
demonstrates an ETB receptor population that mediates little vasoconstriction (Bacon and Davenport, 1996). Therefore,
the right coronary artery is a good model to use to exclusively
study the function (i.e., vasoconstriction and Cam response)
of ETA receptors.
This study uniquely demonstrates that the Cam response
mediated by either the ETA or ETB receptor in isolated
smooth muscle cells parallels the development of isometric
tension in both cold-stored coronary arteries and organ-cultured coronary arteries. Organ culture greatly enhanced both
the Cam response and isometric tension development to ET-1.
Our results indicate that the increased response to ET-1 is
attributed to an enhanced action of ETA receptors, and not
ETB receptors.
Enhanced ETA Receptor Function with Organ Culture