From the Department of Physiology, Medical College of Wisconsin,
Milwaukee. Dr Tiefenbacher is now at the Medizinische Klinik III/Kardiologie,
Universität Heidelberg, Germany. Dr DeFily is now at the Center for
Anesthesiology Research, Cleveland Clinic Foundation, Cleveland, Ohio.
Correspondence to William M. Chilian, PhD, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226. E-mail chilian{at}post.its.mcw.edu
Methods and Results—Administration of the
Conclusions—Cardiac myocytes have a requisite role in
constriction of coronary resistance vessels to
Cardiac myocytes were enzymatically isolated and
purified.17 The left coronary artery of a
dog heart was perfused with collagenase-buffer mix (pH 7.4)
for 30 minutes at 37°C.17 Then the left
ventricle was minced into small, 0.25- to 0.5-g pieces in 100 mL of the
collagenase-buffer solution and further digested in an
orbital shaker (240 min-1) for 20 to 40 minutes
(37°C). This crude fraction was filtered through surgical gauze and
resuspended in buffer. Aliquots of this suspension were placed in 50-mL
conical tubes, and the myocytes were allowed to settle for 10 minutes.
Floating cells and debris were aspirated, and the myocyte fraction was
resuspended, then allowed to settle for 5 minutes. This procedure was
repeated, and myocytes were resuspended in buffer with 1% BSA.
CaCl2 was added incrementally in five 5-minute
steps to a final [Ca2+] of 1 mmol/L.
Myocytes were concentrated to 1.5x107 to
2.0x107/mL by rinsing the suspension through
cheesecloth and collecting the unfiltered fraction, then pipetting it
into centrifuge tubes. Myocytes were maintained at 37°C in a
humidified incubator (20% O2, 5%
CO2, 75% N2) in buffer
containing the following additional compounds (mmol/L): taurine 60,
creatine 20, and CaCl2 1, plus 1.5% BSA. The
purity of the suspension was verified by microscopic analyses.
Viability was evaluated by trypan blue exclusion and a rodlike
configuration after videomicroscopic images were digitized and the
total areas of all and of dead (nonviable) myocytes planimetered. The
area of dead cells was expressed as a percentage of the total area;
thus, 1-% dead cells= % viable myocytes. The average viability of
cells was 91% (range, 82% to 96%) of 14 images from 4 different
experiments).
Endothelin-1 levels were assessed in the myocyte-derived supernatant
under basal conditions and during stimulation by
phenylephrine with a radioimmunoassay (Amersham).
Chemicals and drugs were obtained from the following sources: buffers,
salts, taurine, creatine, and adenosine, Sigma Chemical Co;
albumin (for microvessel preparations) (crystallized bovine,
>98% purity), US Biochemical; albumin (for myocyte
isolation), fraction V, Calbiochem; phenylephrine
hydrochloride, S(-)propranolol hydrochloride,
prazosin hydrochloride, serotonin hydrochloride, sodium
nitroprusside, 8-(p-sulfophenyl)theophylline, and
endothelin-1 (peptide free base), Research Biochemicals International;
collagenase (type 2), Worthington; and endothelin-A
receptor peptide antagonist FR 139317, Abbott Laboratories.
All drugs, except prazosin, used for the studies of vasoactivity were
dissolved in PSS without albumin to make stock solutions (1 to
10 mmol/L), then divided into aliquots and stored at -20°C.
Aliquots were used once and kept on ice during the experiments.
Prazosin was initially dissolved in absolute ethanol to make a 10
mmol/L stock, then diluted 2-fold in PSS. When the prazosin-ethanol was
added to the organ chamber, the final concentration of ethanol in the
microvascular organ chamber was 0.1%, which when added as a vehicle
did not affect tone.
Vasoactive reactions were assessed in coronary arterioles (n=6
to 9) by addition of the drugs directly to the microvessel organ
chamber (2-mL volume) or to the myocyte suspensions, which were
apportioned into 1-mL volumes (1.5x107 to
2.0x107 cells). After 20 minutes of incubation
with the drug, the myocytes were "pelleted" with gentle
centrifugation (500 rpm), and the supernatant was
collected and administered to the isolated arterioles (20-µL
aliquots). All results are expressed as mean±SEM. Percent dilation was
calculated as (diameter during an intervention minus baseline
diameter)/(diameter during maximal dilation with
10-5 mol/L adenosine minus baseline
diameter). Vasoconstriction was calculated as the percent decrease in
diameter from baseline and is expressed as -%. The average baseline
diameter of all vessels was 58±3 µm, and the maximal dilation
was 70±4 µm. Resolution with the x20 objective was 1
µm. Results were analyzed by 1- or 2-way repeated-measures
ANOVA and are presented as mean±SEM. A value of
P<0.05 was accepted for statistical significance.
Administration of the M-D S/N to the microvessel produced vasodilation,
which was attenuated by the adenosine antagonist
8-PSPT (50 µmol/L) (Figure 1
Supernatant (20 µL) from phenylephrine-treated myocyte
suspensions (10-6, 10-5,
and 5x10-5 mol/L) produced less vasodilation as
the dose of phenylephrine was increased (Figure 2
Administration of the
The control level of ET-1 in the supernatant was 66±10 pmol/L, and it
increased to 102±18, 104±12, and 118±12* pmol/L (*P<0.05
versus control) during stimulation with 10-6,
10-5, and 5x10-5 mol/L
phenylephrine, respectively.
Can we eliminate the possibility that our in vitro microvessel
(arteriole) preparations have been injured, rendering them unresponsive
to
What is the physiological significance of
endothelin-1 production by cardiac myocytes? This is a puzzling
aspect of our results. We found that phenylephrine
treatment increased endothelin-1 levels in the myocyte-derived
supernatant to >100 pmol/L. Because the supernatant was diluted
100-fold in the organ chamber, the final concentration of endothelin
would be below threshold to elicit vasoconstriction. Yet, we found that
the constriction induced by the supernatant of
phenylephrine-treated myocytes was blocked by the
ETA antagonist. Our best
reconciliation between these seemingly disparate results is to
hypothesize that during
The present findings also corroborate a preliminary observation we
made; specifically, phenylephrine-induced
coronary vasoconstriction of epicardial coronary
arterioles in situ was inhibited by an ETA
antagonist.22 This observation
provides compelling evidence that our in vitro observations extend to
the intact coronary circulation. Although we cannot state
unequivocally that endothelin-1 is the single causal factor, our
results imply that activation of ETA receptors
may be involved as physiological effectors of
coronary arteriolar constriction during
Received April 3, 1998;
revision received May 5, 1998;
accepted May 6, 1998.
© 1998 American Heart Association, Inc.
Brief Rapid Communications
Requisite Role of Cardiac Myocytes in Coronary
1-Adrenergic Constriction
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Background—-Adrenergic activation
in vivo causes constriction of coronary arterioles, but,
paradoxically, in vitro these microvessels do not contract to this
stimulus. We hypothesized that cardiac myocytes have a requisite role
in 1-adrenergic coronary arteriolar constriction
through the release of myocyte-derived contractile factor(s). 1-adrenergic agonist phenylephrine did not
constrict isolated coronary arterioles, but constriction was
observed to supernatant obtained from phenylephrine-treated
cardiac myocytes. Constriction to the supernatant was blocked by
administration of an endothelin-A antagonist to the
microvessel preparation or an -adrenergic antagonist to
the myocytes and was augmented after administration of an
adenosine antagonist. Administration of
phenylephrine to the myocytes increased endothelin-1 levels
in the supernatant, but only to subthreshold concentrations. 1-adrenergic stimuli, which may be mediated by
endothelin-1 and other unidentified myocyte-derived vasoconstrictors.
Key Words: microcirculation • endothelin • phenylephrine • dogs
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
Numerous
laboratories, including ours, reported that coronary resistance
vessels constrict to -adrenergic
activation,1 2 3 4 5 but we and another group made the
contrasting observation that isolated coronary arterioles are
refractory to -adrenergic agonists.6 7 This is
puzzling, because isolated arterioles from a variety of organ
systems,6 8 9 and in vitro coronary
venules6 and large coronary arteries
(>500 µm in diameter)10 11 12 constrict to
1- and/or
2-adrenergic stimulation. A reconciliation
between in vivo and in vitro coronary arteriolar responses to
-adrenergic activation would occur if arteriolar vasoconstriction in
vivo is produced indirectly, ie, activation of -adrenergic receptors
on parenchymal cells provokes the release of a factor that induces
coronary vasoconstriction. Because our microvessel preparations
are devoid of cardiac myocytes, we hypothesized that these cells have a
requisite role in modulating coronary arteriolar constriction
to 1-adrenergic activation. We tested this by
examining the vasoactive responses of isolated arterioles to graded
doses of the 1-adrenergic agonist
phenylephrine and by reexamining the arteriolar responses
to aliquots of fluid obtained from isolated myocytes treated with
phenylephrine. Autoradiographic studies have
detected a substantially higher density of
1-adrenergic receptors on cardiac myocytes
than on coronary arterioles,13 which adds
substance to our argument that cardiac myocytes are "targeted" by
1-adrenergic agonists. We also proposed that
in vivo, 1-adrenergic agonists induce
coronary arteriolar constriction by stimulating cardiac
myocytes to release endothelin-1, which constricts coronary
resistance vessels. To test this, we assessed vasoactive responses
during antagonism of ETA receptors on arterioles
and 1-adrenergic receptors on cardiac myocytes
and measured endothelin-1 production by cardiac myocytes during
1-adrenergic activation. Although cardiac
myocytes possess 1-adrenergic
receptors14 and produce
endothelin-1,15 a functional link between these
characteristics is not established.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
The use of animals was in accordance with Medical College of
Wisconsin and National Institutes of Health guidelines, and protocols
were approved by the institutional laboratory animal care committee.
Coronary arterioles (50 to 80 µm in intraluminal
diameter) were isolated from the canine hearts (n=11), cannulated with
micropipettes at both ends, bathed in buffered albumin–PSS (pH
7.4, 37±1°C), and studied with
videomicroscopy.16 The arterioles developed
spontaneous tone during equilibration (60 cm
H2O).
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
The 1-adrenergic agonist
phenylephrine administered directly to the microvessel did
not elicit any change in diameter in either the absence or presence of
ß-adrenergic blockade (propranolol
10-6 mol/L). During ß-adrenergic blockade,
phenylephrine at 10-6,
10-5, and 5x10-5 mol/L
produced relaxation of 4±1%, 9±1%, and 10±1%, respectively (n=8).
The vessels constricted (20% to 40% decrease in diameter) to
endothelin-1 (10-10 mol/L) and dilated near
maximally (20% to 30% increase in diameter) to
endothelium-independent (adenosine,
10-6 mol/L; nitroprusside,
10-5 mol/L) and
endothelium-dependent (serotonin,
10-5 mol/L) agonists, indicating that they were
responsive to several agonists. Responses to ET-1 were not completed in
every vessel because the vasoconstriction often lasted for 90 to 120
minutes, but we evaluated constriction in 5 vessels to
10-10 mol/L endothelin (-29±4%) and completed
a full dose-response relationship to endothelin-1 (n=3;
10-12 mol/L, -6%; 10-11
mol/L, -18%; 10-10 mol/L, - 29%;
10-9 mol/L, -48%; and
10-8 mol/L, -65%). 
).
This dose of 8-PSPT shifted the dose-response curve to
adenosine by 2 log orders (data not shown). These results
suggest that the isolated cardiac myocytes are producing vasodilatory
quantities of adenosine.
View larger version (20K):
[in a new window]
Figure 1. Effects of myocyte-derived supernatant obtained
under control conditions (control; n=8) on arteriolar dilation and
administered after adenosine antagonism with 50 µmol/L
8-PSPT (n=8), which was added to microvessel chamber (refer to inset
showing route of administration of substance; this format will be used
in subsequent figures). Dilation to myocyte-derived supernatant was
significantly attenuated by 8-PSPT. Baseline diameters were 58±3
µm (control) and 58±3 µm (8-PSPT). ). Administration of 8-PSPT to the
microvessel preparation unmasked dose-dependent vasoconstriction of the
supernatant from the phenylephrine-treated myocytes (Figure 2). The constriction lasted for >90 minutes. The constricted
microvessels dilated maximally to serotonin
(10-5 mol/L) or nitroprusside
(10-5 mol/L), demonstrating that the progressive
constriction was not due to deterioration of the preparations.
View larger version (23K):
[in a new window]
Figure 2. Comparison of vasoactive responses of isolated
arterioles to 20 µL M-D S/N from controls (control; n=9), myocytes
treated with 3 different doses of phenylephrine (n=9) added
to myocyte suspension, or myocytes treated with doses of
phenylephrine and then administered to microvessels with
8-PSPT (phenylephrine+8-PSPT; n=9).
Phenylephrine treatment inhibited vasodilatory properties
of M-D S/N (vs control), and after administration of 8-PSPT, it induced
vasoconstriction. Baseline diameters were control, 69±5 µm;
phenylephrine, 70±6 µm; and
phenylephrine+8-PSPT, 64±5 µm. 1-adrenergic
antagonist prazosin (10-6 mol/L) to
the myocyte suspension before administration of
phenylephrine inhibited the production of
contractile factor(s) by the cardiac myocytes (Figure 3). Prazosin given directly to the
microvessels did not alter the vasoconstrictive
properties of the supernatant (n=6). The ETA
antagonist FR 139317, 10 µmol/L, given to the
arteriole blocked the vasoconstrictive properties of
the supernatant from phenylephrine-treated cardiac myocytes
(Figure 3). This dose of antagonist blocked
vasoconstriction to 100 pmol/L endothelin-1 but did not alter baseline
tone or affect vasodilation to adenosine or
serotonin, suggesting antagonist specificity at
this dose.
View larger version (24K):
[in a new window]
Figure 3. Vasoactive effects of M-D S/N (20 µL) in vessels
treated with 8-PSPT. M-D S/N was obtained from myocytes treated with
phenylephrine (phenylephrine+8-PSPT; n=9),
myocytes simultaneously treated with
phenylephrine and prazosin
(phenylephrine+prazosin+8-PSPT; n=6), or myocytes treated
with phenylephrine and with ETA
antagonist administered to microvessel
(phenylephrine+FR 139317+8-PSPT; n=4). Baseline diameters
were phenylephrine+8-PSPT, 65±5 µm;
phenylephrine+prazosin+8-PSPT, 72±5 µm; and
phenylephrine+FR 139317+8-PSPT, 57±6 µm.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
We have found that cardiac myocytes exert a requisite role in
1-adrenergic coronary arteriolar
constriction. The results also suggest that the coronary
constriction is mediated, in part, by endothelin(s), because the
ETA antagonist blocked the
constrictor properties of the supernatant. However, we cannot make
unequivocal assertions about the role of endothelin-1, because its
concentration in the supernatant from phenylephrine-treated
myocytes would be below threshold after the dilution of the supernatant
in the bath (1:100) is considered. It is important to highlight some
considerations that are pertinent to our results and conclusions. 1-adrenergic activation? The failure of
isolated arterioles is not related to a more general problem with
receptor coupling. Agonists signaling through G
protein–coupled receptors include
1-adrenergic agonists,18
angiotensin II,19
endothelin-1,20 and U44619
(thromboxane mimetic),21 and
coronary arterioles directly respond to all of these factors
except 1-adrenergic agonists. Furthermore,
isolated coronary venules and skeletal muscle arterioles
constrict during 1- or
2-adrenergic activation, demonstrating that
our experimental procedures do not eliminate these adrenergic
responses.6 We believe that our preparations have
not produced an artifact but rather that isolated coronary
arterioles do not respond directly to
1-adrenergic activation. 1-adrenergic
activation, cardiac myocytes are producing constrictors other than
endothelin-1. It is possible that other endothelins that would be
blocked by ETA antagonist are
produced by the myocytes. Alternatively, endothelin-1 could have a
"permissive" effect on or synergize the action of another
vasoconstrictor. Even though our dissimilar results preclude an
unequivocal conclusion about the role for endothelin-1 in mediating the
constriction, we believe that our most important observation is that
cardiac myocytes have a requisite role in mediating
1-adrenergic coronary arteriolar
vasoconstriction. 1-adrenergic activation of cardiac myocytes.
We conclude with conviction that cardiac myocytes exert a requisite
role in 1-adrenergic constriction of
coronary resistance vessels.
Selected Abbreviations and Acronyms
ETA
=
endothelin-A receptors
M-D S/N
=
myocyte-derived supernatant
8-PSPT
=
8-(p-sulfophenyl)theophylline
PSS
=
physiological salt solution
Acknowledgments
This work was supported by NIH grants HL-32788 and HL-51748
(W.M.C.), the German Research Foundation (C.P.T.), and the American
Heart Association, grant 94-15050 (D.V.D.). We acknowledge the gift of
FR139317 from G.D. Searle. We thank Francois Abboud, Allen Cowley,
William Campbell, Eric Feigl, and Julian Lombard for their critical
review of this paper.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1- and 2-adrenergic
receptors in the coronary microcirculation.
Circulation. 1991;84:2108–2122.
1- and 2-adrenergic
constriction in the coronary microcirculation.
Circulation. 1993;87:1264–1274.
1- and
2-adrenoceptor-mediated vasoconstriction of
large and small canine coronary arteries in vivo.
J Cardiovasc Pharmacol. 1984;6:961–968.[Medline]
[Order article via Infotrieve]
-Adrenergic responses of isolated canine coronary
microvessels. Basic Res Cardiol. 1995;90:61–69.[Medline]
[Order article via Infotrieve]
-adrenergic activation elicits endothelin-induced arteriolar
vasoconstriction. Circulation. 1995;92(suppl I):I-321.
Abstract.
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