Download Endothelium-Derived Relaxing Factor in the

123
Mechanical Deformation of Vessel Wall and
Shear Stress Determine the Basal Release of
Endothelium-Derived Relaxing Factor in the
Intact Rabbit Coronary Vascular Bed
Daniel Lamontagne, Ulrich Pohl, and Rudi Busse
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We investigated the mechanisms that are responsible for the basal release of endotheliumderived relaxing factor (EDRF), which is likely to be identical with nitric oxide, in the intact
coronary circulation. The increase in cGMP content of platelets passing through the coronary
bed of the isolated rabbit heart was used as an index of EDRF release. Platelet cGMP content
after passage through the heart under control conditions (flow rate of 20 ml/min) amounted to
0.50±0.10 pmol/mg protein. Inhibition of endothelial nitric oxide synthesis by 30 ,uM
NG-nitro-L-arginine (L-NNA) reduced this amount by more than 60%. Increasing flow rate from
20 ml/min to 40 and 60 ml/min led to flow-dependent dilation as reflected by the subsequent
drop in perfusion pressure after an initial rise. The flow-dependent dilation was associated with
a significant increase in the normalized platelet cGMP content. L-NNA abolished completely
both the flow-dependent dilation and the increase in platelet cGMP content. Increasing shear
stress by a strong vasoconstriction (1 nM endothelin-1) at constant flow was also accompanied
by a 2.5-fold increase in platelet cGMP content. To investigate whether mechanical forces
applied to the vascular wall by the myocardial contraction cycle were also a stimulus for EDRF
release, cardiac arrest was induced by a continuous infusion of mepivacaine (final concentration, 0.02%). Under these conditions, a decrease in platelet cGMP content comparable to that
after nitric oxide synthesis inhibition was observed in the arrested heart. The reappearance of
mechanical activity after washout of mepivacaine was associated with the recovery of platelet
guanylate cyclase activation, indicating the contribution of the mechanical activity on basal
EDRF release. To study further the effects of wall deformation on EDRF release, experiments
were performed in isolated saline-perfused rabbit arterial segments. Rhythmic squeezing of the
segments led to a significant increase in EDRF release as assessed by assay of guanylate cyclase
activity. These data indicate that at least two principles contribute to the basal agonistindependent release of EDRF in the intact coronary bed. Besides the continuously acting wall
shear stress imposed by the streaming fluid, the periodic compression of intramyocardial
vessels stimulates the formation of EDRF. This EDRF formation may result from the high
shear stress imposed on the endothelial lining by the periodic diameter reduction and from the
direct deformation of the endothelium. (Circulation Research 1992;70:123-130)
The endothelium-derived relaxing factor
(EDRF), which is likely to be identical with
nitric oxide (NO),1,2 can be released after the
action of several blood-borne agonists on their specific receptors.3 On the other hand, several studies
From the Institute of Applied Physiology, University of
Freiburg, Freiburg, FRG.
Supported by a grant from the Deutsche Forschungsgemeinschaft (Bu 436/4-1 and 4-2). D.L. is a Research Fellow of the
Heart and Stroke Foundation of Canada.
Address for correspondence: Rudi Busse, Institut fiir Angewandte
Physiologie, Hermann-Herder-Str. 7, D-7800 Freiburg, FRG.
Received February 21, 1991; accepted September 16, 1991.
support the concept that the intact coronary vascular
bed is maintained under a constant dilating tone by a
basal, agonist-independent release of EDRF. A significant amount of EDRF is released from the isolated guinea pig45 and rabbit6 heart without any
agonist stimulation. Furthermore, inhibition of NO
synthesis is accompanied by a considerable vasoconstriction in the coronary vascular bed of the isolated
rabbit heart.7 8 However, the stimuli or mechanisms
responsible for such a basal release of EDRF are still
unclear.
The release of EDRF can be stimulated by shear
stress in isolated arterial segments9 and cultured
124
Circulation Research Vol 70, No 1 January 1992
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endothelial cells.10 Although the shear stress-induced release of EDRF in the intact coronary microcirculation has yet to be confirmed, the continuous
shear stress imposed on the endothelial lining by the
streaming fluid in the coronary vascular system could
theoretically stimulate the basal, agonist-independent release of EDRF. Endothelial cells are also
stimulated by the deformation of the vessel wall. For
example, pulsatile flow, which is accompanied by
rhythmic changes in vessel diameter, is a more potent
stimulus of EDRF release than nonpulsatile flow.11
Since the coronary vascular bed is constantly exposed
to the rhythmic compression and decompression
associated with the contraction-relaxation cycle of
the myocardium, the mechanical activity of the beating heart could also stimulate the release of EDRF,
therefore contributing to the basal release of EDRF
in this organ.
This study was thus performed to assess the contribution of shear stress and the cardiac mechanical
activity as stimuli for the basal, agonist-independent
release of EDRF in the intact coronary vascular bed
of the isolated rabbit heart.
Materials and Methods
Isolated Perfused Rabbit Hearts
Mongrel rabbits (1.0-1.5 kg) of either sex were
anesthetized with sodium pentobarbital (30-60
mg/kg i.v.). After anticoagulation with heparin (1,000
IU i.v.), the carotid artery was transected, and the
heart was rapidly perfused in situ (Langendorff preparation) through a cannula inserted into the aortic
stump. The heart was then excised and perfused at a
constant flow rate by means of a roller pump
(Heidolph RGL 85, Kelheim, FRG). Two compliance chambers along the perfusion line ensured a
continuous flow. The perfusion solution consisted of
a modified Krebs-Henseleit buffer containing (mM)
NaCl 118, KCI 4, CaCI2 2.5, KH2PO4 1.2, MgSO4 1,
NaHCO3 24, D-glucose 5, and pyruvate 2, dissolved in
double-distilled water. The perfusate was gassed with
95% 02-5% CO2 (pH 7.4) and kept at a constant
temperature of 37°C. Bolus doses of drugs and
platelets were administered through a Y connector in
the aortic perfusion line. Coronary perfusion pressure (CPP) was measured with a pressure transducer
(model P2310, Gould, Oxnard, Calif.) connected to a
side arm of the aortic perfusion cannula. Isovolumetric left ventricular pressure was measured by a fluidfilled latex balloon inserted into the left ventricle
through a pulmonary vein and connected to a second
pressure transducer (model CP-01, Gould). The volume of the balloon was adjusted to obtain a diastolic
pressure between 5 and 10 mm Hg. Fluid that could
enter the ventricle was drained by means of a vent in
the apex of the left ventricle. Heart rate was derived
from the left ventricular pressure signal by a cardiotachometer. The coronary effluent was collected
from the coronary sinus and the right atrium. Passage
through the right ventricle was prevented by destruc-
tion of the atrioventricular and pulmonary valves and
ligation of the pulmonary arterial trunk.
Platelet Preparation
Blood from healthy human donors (70-80 ml) who
had not received any medication was collected in a
3.8% sodium citrate solution (10% of final volume).
Platelet-rich plasma was obtained by centrifugation
at 200g for 20 minutes. After addition of citric
acid/sodium citrate/dextrose (20 vol%) and prostacyclin (75 pg/ml), the platelet-rich plasma was centrifuged again (800g), and the pellet was suspended in a
modified HEPES-buffered Tyrode's solution containing indomethacin (10 ,uM) to obtain a final platelet
count of 2-3 x 106 platelets/gl. Prostacyclin was eliminated from this suspension by a final 10-minute
incubation at 37°C.
Experimental Protocols
The basic experimental protocol consisted of evaluating the cGMP increase in platelets passing
through the coronary bed, as an index of luminally
released EDRF, under unstimulated conditions and,
10 minutes later, during endothelial stimulation with
1 ,uM acetylcholine (ACh, Sigma Chemical Co.,
Deisenhofen, FRG). This protocol was performed
using different flow rates and pharmacological interventions. After a first stabilization period (15-20
minutes), each experiment started with a test response to the endothelium-dependent dilator ACh,
followed by a further 30-minute stabilization period.
The platelet cGMP content was evaluated with pairs
of perfusate samples: a platelet-free sample taken 30
seconds before platelet injection and a sample containing the collected platelets. Platelets (300-gl
bolus) were injected at time 0, over a 2-second
period, and collected between 4 and 8 seconds, 3 and
5 seconds, and 2 and 3.5 seconds for perfusion rates
of 20, 40, and 60 ml/min, respectively. An infusion of
0.1 ,uM superoxide dismutase (SOD, Serva, Heidelberg, FRG) was started 2 minutes before each bolus
injection and maintained until platelets had been
collected. In all experiments, the initial perfusion
rate was set at 20 ml/min and, unless otherwise
stated, maintained constant.
In a first experimental series (n =7), bolus injections
of platelets were administered before and after inhibition of endothelial NO synthesis (30-minute infusion of 30 ,uM N0-nitro-L-arginine [L-NNA], Serva).
To assess whether platelet-derived NO12 contributes
to cGMP increases, platelets were incubated for 30
minutes with 100 ,uM L-NNA in additional experiments (n=3). In a second experimental series (n=6),
the effect of flow rate on NO release was studied. NO
release was determined at perfusion rates of 20, 40,
and 60 ml/min under unstimulated conditions and
during ACh stimulation at 3 and 13 minutes after the
flow increase, respectively. In a third experimental
series (n = 8), we studied the effect of increasing shear
stress on NO release at a constant flow rate by means
of a vasoconstriction induced by a 1-nM infusion of
Lamontagne et al Mechanisms of Basal EDRF Release
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endothelin-1 (Peninsula Laboratories, Inc., Belmont,
Calif.). In a final experimental series (n = 13), luminal
NO release was determined before and 10 minutes
after the cardiac arrest induced by a 0.02% mepivacaine (Astra Chemical, Wedel, FRG) infusion and
again after washout of this drug.
Platelet cGMP Content
The coronary effluent collected was pipetted into
tubes containing 100% trichloroacetic acid (final
concentration of 6%). After centrifugation, trichloroacetic acid was extracted from the supernatant with
water-saturated diethyl ether (four times, 5 vol ether
for 1 vol sample), and the samples were kept at
-20°C until analysis. cGMP was determined using a
commercially available radioimmunoassay (New England Nuclear, Dreieich, FRG). The platelet cGMP
content was calculated from the difference between
the platelet-containing and platelet-free samples and
was expressed as picomoles of cGMP per milligram
of platelet protein.
Protein Content
The pellet of the centrifuged coronary effluent
samples was redissolved in 100 gl of 1N NaOH, and
the protein content of the sample was determined
according to Lowry et al.13
Infusion of Drugs
ACh, L-NNA, and SOD were dissolved in physiological perfusion buffer and infused at ¼loo of the
coronary flow rate to produce a final concentration of
1, 30, and 0.1 ,M, respectively. Endothelin-1 was
dissolved in a 0.2% bovine serum albumin solution
and diluted with physiological perfusion buffer for a
final concentration of 1 nM. Mepivacaine (Scandicain 1% solution, Astra Chemical) was infused at 2/oo
of the coronary flow rate to produce a final concentration of 0.02%.
NO Release From Isolated Perfused Arterial Segments
Segments of rabbit femoral arteries (1.5 cm in
length) with an intact endothelium were freed from
adventitial tissue, cannulated at both ends, and
placed in an organ bath. Luminal perfusion (10.4
ml/hr) was performed separately from organ bath
perfusion with normal Tyrode's solution. The shear
stress on the endothelium was increased by reducing
the vessel diameter with either 30 ,uM norepinephrine (Arterenol, Hoechst AG, Frankfurt, FRG) or 60
nM endothelin-1, while maintaining the same perfusion rate. The effect of mepivacaine was tested under
these experimental conditions to assess its effect on
shear stress-induced release of EDRF. The effect of
mechanically induced vessel wall deformation on
EDRF release was also studied in the isolated perfused arterial segments. Two opposing plates, covered with a soft material, were used to perform a
rhythmic squeezing (90 per minute) of the segments,
with the lumen practically closed during the compression phase.
-
2.U l1
1
=
125
control
m ACh
L._
0
0.
c,
*
1.5-
E
E 1.0_.
0c
0.5-
0
4)
-6J
0.0 -
without
L-NNA
L-NNA
L-NNA
(endothelium) (platelets)
FIGURE 1. Bar graph showing platelet cGMP content after
passage through the coronary bed under control conditions
and during acetylcholine (ACh, 1 pM) infusion, both in the
absence of NG-nitro-L-arginine (L-NNA) (n= 7), after a 30minute intracoronary infusion of 30 MM L-NNA (n= 7), or
after a 30-minute preincubation of platelets with 100 ,uM
L-NNA (n=3). *p<0.05.
The EDRF released from these segments was
evaluated by activation of purified guanylate cyclase
(GC) from bovine lungs.14 Effluent from the perfused
vessels was collected for 15 seconds into a reaction
mixture containing triethylamine-HCl buffer (30
mM, pH 7.4), MgCl2 (3 mM), SOD (0.3 ,uM), 0.1 mM
[a-32P]GTP (0.2 ,uCi), glutathione (3 mM), EGTA
(0.1 mM), bovine gamma globulin (0.1 mg/ml), and
soluble GC (200 nM) purified to apparent homogeneity. The reaction proceeded for 1 minute at 37°C
and was terminated by the addition of 50 mM zinc
acetate and 55 mM sodium carbonate. Labeled
cGMP was then separated from other nucleotides on
small alumina columns, and GC activity was calculated. A detailed description of this technique is
given elsewhere.15
Statistics
Heart rate and CPP were analyzed with parametric
statistical tests. A paired Student's t test was used for
comparison of two observations; an analysis of variance for repeated measurements was used for three
or more observations. Since platelet cGMP data were
not normally distributed, nonparametric tests were
used. A Wilcoxon matched-pair signed-rank test was
used to compare two paired observations; a Friedman test followed by a Wilcoxon and Wilcox multiple
comparison was used for more than two observations.
All results are presented as mean+SEM.
Results
Effect of L-NNA on Platelet cGMP
The cGMP content of platelets before passage
through the heart amounted to 0.18+±0.04 pmol/mg
protein (n =9). When platelets were injected into the
coronary bed, the cGMP content increased by 2.8fold under control conditions and by 7.5-fold during
ACh stimulation (Figure 1). After a 30-minute infu-
126
Circulation Research Vol 70, No 1 January 1992
9: from 40
to 60 mI/min
A: from 20
to 40 ml/min
150
G)
Q)
60-
c
-
0
a)(L)
Q} 100
E
E
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40
-
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00
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.
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.
.
.
.
.
20
.
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(0
5
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time [min]
FIGURE 2. Coronary perfusion pressure (CPP) plotted as a
function of time after increases in perfusion rate from 20 to 40
mllmin (panel A) and from 40 to 60 ml/min (panel B).
Horizontal error bars represent ±SEM of the time after which
the minimal pressure value was observed. *p<0.05 (n=6)
compared with the preceding value.
0
60
40
20
flow rate [mi/min]
FIGURE 3. Bar graph showing platelet cGMP content, expressed as percent of the platelet cGMP content measured
during 1 iM acetylcholine (ACh) infusion, under different
perfusion rates. *p<O0.05 (n=6).
sion with L-NNA, the cGMP content of platelets
passing through the unstimulated heart (0.18±0.08
pmol/mg protein) was similar to the amount found in
platelets before passage, indicating that a basal,
agonist-independent EDRF release was responsible
for the increase in platelet cGMP content under
control conditions. Likewise, the ACh-induced increase in platelet cGMP was totally blocked by
L-NNA (Figure 1). In contrast, platelets treated with
100 ,uM L-NNA showed a similar increase in cGMP
after passage through the heart under control conditions as well as during ACh stimulation (Figure 1),
which excludes any important contribution of platelet-derived NO in the cGMP content increases we
observed in our experimental conditions.
Effect of Increases in Flow Rate
Increases in flow rate from 20 to 40 ml/min and
from 40 to 60 ml/min were accompanied by proportional increases in CPP and led to flow-dependent
dilation, as reflected by the significant drop in CPP
that followed the initial rise (Figure 2).
To detect the effect of increasing flow on luminal
NO release, one has to consider that the platelet
transit time is inversely related to the flow rate.
Consequently, the platelet cGMP content under ACh
stimulation decreased linearly with increasing flow
rate (2.52±0.61, 1.31+0.66, and 0.78±0.37 pmol/mg
protein for 20, 40, and 60 ml/min, respectively).
Expressed in percent of the values obtained during
ACh stimulation, the platelet cGMP content under
unstimulated conditions showed a significant increase with increases in flow rate (Figure 3).
The effect of NO synthesis inhibition with L-NNA
(30 ,M) on the flow-dependent dilation was studied
in a separate series of experiments. A 30-minute
infusion of L-NNA reversed the ACh-induced vasodilation to a constriction and prevented the flowdependent dilation (Figure 4). The dilator response
gM sodium nitroprusside infusion was not
affected by L-NNA. None of these effects were
observed with the inactive D stereoisomer of L-NNA.
Effect of an Increased Shear Stress Induced
by Vasoconstriction
The strong vasoconstriction produced by endothelin-1 in the coronary vascular bed was used to increase shear stress while maintaining constant flow
rate. Infusion of 1 nM endothelin-1 increased the
CPP from 37±3 to 101±7 mm Hg (p <0.001). Under
these conditions, the cGMP content of platelets
passing through the vascular bed was increased 2.5fold (Figure 5). Despite the strong vasoconstriction,
the dilator response to ACh (20±6% before versus
24±4% during infusion of endothelin-1) as well as
the ACh-induced increase in platelet cGMP (Figure
to 1
ACh
130- -
flow increase (2 fold)
CPP
[mm3Hg]
30J
.-NHA
W 1 min
FIGURE 4. Original recordings of coronary perfusion pressure (CPP) during an infusion of 1 1iM acetylcholine (ACh)
and after a twofold increase in perfusion rate (from 25 to 50
ml/min), both before and after a 30-minute infusion with 30
/gLM NG-nitro-L-a,ginine (L-NNA). Similar results were obtained in five additional experiments.
Lamontagne et al Mechanisms of Basal EDRF Release
A: unstimulated heart
1.5*
1.0o
2
C
.0
0
0
0.
-U,
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cr
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1.0
CL
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A: unstimulated heart
B: ACh-stimulation
127
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control
endothelin
control
endothelin
Downloaded from http://circres.ahajournals.org/ by guest on May 4, 2017
FIGURE 5. Bar graphs showing platelet cGMP content after
passage through the coronaty bed of the unstimulated heart
(panel A) and the increase in platelet cGMP content after the
induction of acetylcholine (ACh, 1 pM) (panel B), both
before (control) and during the strong vasoconstriction induced by infusion of endothelin-1 (1 nM). *p<0.05 (n=8).
5) were not affected. In this group, a significant
positive correlation between platelet cGMP content
and CPP was observed (Figure 6, top panel), indicating that NO release is proportional to shear stress.
Effect of Cardiac Arrest Induced by Mepivacaine
Heart rate and developed left ventricular pressure
under control conditions were 157±8 beats per
2.4
r=0.63, p<0.05
* control
o endothelin
i
-
,'
.I
1.6
- 00
._
0-m
0.8
cL-
0.0
j
5-
80
40
CPP
Q-
120
[mmHg]
0
0~
CD
0.6
r=0.09, ns
0
0a
.:4
.0
0.4~
0*
0
CL
0.2
*
*
0
L
*B
0.0
l00
150
200
heart rate [beats/min]
FIGURE 6. Scatter plots showingplatelet cGMP content as a
function of coronary perfusion pressure (CPP) (top panel)
and of heart rate (bottom panel). Linear regressions (solid
line) are shown along with the 95% confidence interval
(dashed lines) in the top panel.
washout
control
mepivocoine
washout
mepivocoine
control
FIGURE 7. Bar graphs showing platelet cGMP content after
passage through the coronary bed of the unstimulated heart
(panel A) and the increase in platelet cGMP content after the
induction of acetylcholine (ACh, 1 MM) (panel B), both
before (control) and during cardiac arrest induced by a 0. 02%
mepivacaine infusion and after washout of this drug. *p<0.05
and **p<0.01 (n=13).
minute and 88 ± 6 mm Hg, respectively. Perfusion of
mepivacaine resulted in a rapid cardiac arrest, with
undetectable levels of developed left ventricular
pressure within seconds. The CPP response to mepivacaine was biphasic, with a rapid pressure drop
(from 40±4 to 28±3 mm Hg, p<0.01) preceding a
slow increase in perfusion pressure (maximal value of
80+3 mm Hg,p<0.01). The cGMP content of platelets passing through the arrested heart was significantly reduced by more than 50%, compared with
that under control conditions (Figure 7). In contrast,
the platelet cGMP content (Figure 7) and the reduction in CPP (21±6% versus 20±3%) during ACh
infusion were not affected by mepivacaine.
Washout of mepivacaine resulted in a rapid recovery of the mechanical activity in all hearts studied:
heart rate was restored to 132±7 beats per minute
(p<0.01 compared with control), and developed left
ventricular pressure was restored to 80±4 mm Hg
(p>0.05). The platelet cGMP content after passage
through the heart was no longer reduced (Figure 7).
The ACh-induced reduction in CPP (26±4%) and
the increase in platelet cGMP (Figure 7) were
slightly increased after washout of mepivacaine, although the difference was not statistically significant.
Mepivacaine modified the cGMP content neither of
resting platelets nor of platelets stimulated in vitro
with sodium nitroprusside (1 and 10 ,uM) (data not
shown).
In a separate experimental series, the effect of an
enhanced heart mechanical activity on platelet
cGMP content was studied with a continuous norepinephrine infusion (1 ,uM). Increases in both heart
rate and contractility by more than 50% did not
significantly affect platelet cGMP content after passage through the coronary system (from 0.29+16 to
0.32±0.19 pmol/mg protein, n=6). Similarly, we
found no significant correlation between platelet
cGMP content and heart rate of unstimulated, spontaneously beating hearts under control conditions
(Figure 6, bottom panel).
128
Circulation Research Vol 70, No 1 January 1992
Release of EDRF (NO) From Isolated
Femoral Segments
At a resting diameter of 1,920±100 ,um, the activity of purified GC added to the effluent from the
segments was 5.3+1.4 nmol* min-l mg-1 protein
(n=6). Both endothelin-1 and norepinephrine induced a strong constriction (1,170±70 ,um) at constant perfusion rate that was associated with an
increase in GC activity to 9.3±2.0 nmol * min-1 mg`
(p<0.01, n=7). The increased EDRF release was no
longer observed when the vasoconstriction was abolished by forskolin (3 gM) (data not shown). The
addition of 0.02% mepivacaine in the luminal perfusion had no influence on the diameter of maximally
constricted femoral segments and did not affect the
shear stress-induced release of EDRF (GC activity,
10.8±2.4 nmol min-l *mg-1).
The rhythmic compression and decompression
(1.5 Hz) of femoral segments also induced an
increase in GC activity from 7.3±0.4 to 17.4±1.0
nmol* min-l mg-1 (p<0.05, n=3). The dilator response to ACh (1 ,uM) was not affected by this
rhythmic compression and decompression (data not
shown), indicating the preservation of the functional integrity of the endothelium.
-
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Discussion
In the present study, we used human platelets as a
bioassay tissue for the detection of EDRF (NO)
released from the luminal side of the coronary vascular bed. Human platelets contain considerable
amounts of soluble GC,16 which can be activated by
NO during a single passage through the coronary
bed. This approach has already been proven to be a
reliable index of EDRF (NO) release.6,8 It has been
reported recently that human platelets stimulated
with collagen can synthesize NO.12 However, it is
unlikely that platelet-derived NO contributes in a
major part to the cGMP increase we observed, since
the preincubation of platelets with a high concentration of L-NNA did not alter the increases in cGMP.
Increments in flow induced a vasodilation in the
coronary bed, as reflected by the pressure drop that
follows the sharp initial rise in CPP. Flow-dependent
dilation has been well documented in epicardial
coronary arteries of laboratory animals'7,18 and humans,1920 as well as in other conduit arteries.2122
Furthermore, it was found to be endothelium dependent.1921,22 Our data indicate that flow-dependent
dilation takes place not only in large conduit arteries
but also in resistance-sized vessels. Increments in
flow were also accompanied by increases in EDRF
release. An enhanced nitrite outflow from the unstimulated rabbit heart induced by an increase in
flow rate has recently been described.23 Our study
adds further evidence of a flow-induced EDRF release from the unstimulated heart. These findings,
added to the fact that L-NNA abolishes the flowdependent dilation, are strongly in favor of EDRF as
the endothelial mediator of flow-dependent dilation
in the coronary bed.
Flow exerts its effect through the shear stress
imposed on the endothelial lining. This is illustrated
best by the experiments with endothelin-1 at constant
flow rate. Endothelin-1 elicited a strong and sustained vasoconstriction that increased both shear
stress on endothelial cells and release of EDRF.
Increasing shear stress in isolated arterial segments
resulted also in an enhanced release of EDRF. There
are reports indicating that endothelin-1 may stimulate directly the release of EDRF from arterial
segments of rats24 and rabbits.25 In rabbit femoral
arteries, however, no EDRF release by endothelin-1
was observed when the constriction was abolished by
a strong vasodilator. This is in agreement with our
previous report in which endothelin-1 did not elicit
EDRF release from rabbit aortic segments.26
In the rabbit heart, bolus injections of endothelin
do not induce vasoconstriction unless hemoglobin is
present or the endothelium is destroyed.27 This can
be interpreted as a direct stimulatory effect of endothelin on endothelial EDRF release. On the other
hand, one cannot rule out the possibility that the
shear stress-induced EDRF release readjusts vessel
tone, maintaining a constant CPP despite the transient exposure of the coronary vessels to the peptide.
Likewise, endothelin has been reported to stimulate
the release of prostacyclin from the isolated rabbit
heart.28 Since flow increases are also accompanied by
an enhanced release of prostacyclin,23 it is possible
that the endothelin-induced release of prostacyclin is
in fact the consequence of an increased shear stress.
Thus, discrimination between direct and shear
stress-induced effects of endothelin is difficult. In
our study, however, the positive correlation between
platelet cGMP and CPP is highly suggestive of a
shear stress-induced release of EDRF.
The platelet cGMP content after passage through
the coronary bed of the unstimulated heart is significantly reduced after endothelial NO synthesis inhibition with L-NNA. This strongly suggests that, under basal conditions, the endothelium continuously
releases EDRF in a concentration high enough to
activate the platelet GC during a single passage
through the coronary system. Other studies have
confirmed the existence of a basal, agonist-independent release of EDRF.4-6 The residual platelet
cGMP content after L-NNA, which is very close to
the cGMP content of resting platelets (before passage through the heart) might reflect their basal GC
activity.
Cardiac arrest induced by mepivacaine led to a
significant reduction in the cGMP content of platelets after passage through the unstimulated heart,
which was reversed after reappearance of heart
mechanical activity. The normal cardiac activity is
accompanied by the cyclic compression and decompression of blood vessels, including endothelial cells.
This mechanical activity, which also imposes a highly
oscillatory flow in the coronary bed, is no longer
Lamontagne et al Mechanisms of Basal EDRF Release
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present during cardiac arrest. Therefore, it is very
likely that the beating heart, by imposing a compression-decompression cycle on the endothelial lining
and by creating an oscillatory shear stress, stimulates
a continuous release of EDRF. The stimulatory
effect of rhythmic endothelial compression and decompression on EDRF release was confirmed in
isolated arterial segments. The similar reduction in
platelet cGMP content with both cardiac arrest and
endothelial NO synthesis inhibition by L-NNA suggests that the mechanical activity is the basal stimulus
for a continuous release of EDRF in the coronary
system. Since increases in heart rate and contractility
do not result in a further increase in platelet cGMP
content, it is likely that this mechanically induced NO
release is already completely activated in the spontaneously beating heart. The lack of a positive correlation between heart rate and NO release is also in
favor of an all-or-none stimulatory effect of the heart
mechanical activity. Moreover, the endothelial lining
of the vascular system is constantly exposed in vivo to
shear stress exerted by the streaming blood. We
found that flow stimulates in a proportional manner
NO formation in the coronary system, superimposed
on the mechanically induced NO release. This flowinduced NO release may then contribute to the
adjustment of the conductivity of conduit and resistance-sized arteries in the heart, by counteracting
neuronal or myogenic vasoconstriction. It is interesting to note that cardiac arrest was accompanied by a
vasoconstriction, most likely due to the absence of
mechanical work and the subsequent reduced production of vasoactive metabolites (such as adenosine
and protons). Despite the resulting increase in shear
stress, a reduction in platelet cGMP content after
passage through the unstimulated arrested heart was
observed, which suggests that, in the saline-perfused
coronary microcirculation, the stimulation of the
endothelium by shear stress is subthreshold with the
absence of the heart mechanical activity.
It has been reported that endothelium-dependent
dilations are reduced by some local anesthetics,
suggesting an inhibition of EDRF release by these
drugs.29 However, we found that mepivacaine has no
inhibitory action on ACh-induced release of EDRF
or on the dilator response to this agonist. We were
also unable to detect an effect of mepivacaine, at the
concentration used in this study, on shear stressinduced release of EDRF in isolated arterial segments. Furthermore, mepivacaine has no effect on
soluble GC of platelets or on the activation of this
enzyme by NO, since neither the basal values nor
sodium nitroprusside-induced increases in cGMP
were modified in the presence of this drug.
In conclusion, flow, by inducing shear stress on the
endothelial lining, stimulates the release of EDRF,
which accounts for the flow-dependent dilation observed in the coronary bed. However, the basal
stimulus for the continuous EDRF formation in the
intact coronary microcirculation may be the mechanical activity of the heart, by imposing an oscillatory
129
shear stress and a direct deformation of blood vessel
wall. In addition to this continuous EDRF formation,
shear stress-induced EDRF release can adjust vascular conductivity of the coronary bed.
Acknowledgments
The skillful technical assistance of H. Miinzel, I.
Winter, and C. Kircher is gratefully acknowledged.
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KEY WORDS * endothelium-derived relaxing factor * endothelium
* flow-dependent dilation * L-arginine analogues * cGMP
platelets
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Mechanical deformation of vessel wall and shear stress determine the basal release of
endothelium-derived relaxing factor in the intact rabbit coronary vascular bed.
D Lamontagne, U Pohl and R Busse
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Circ Res. 1992;70:123-130
doi: 10.1161/01.RES.70.1.123
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