Mechanisms responsible for the heterogeneous coronary microvascular response to
nitroglycerin.
M A Kurz, K G Lamping, J N Bates, C L Eastham, M L Marcus and D G Harrison
Circ Res. 1991;68:847-855
doi: 10.1161/01.RES.68.3.847
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 1991 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/68/3/847
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/
Downloaded from http://circres.ahajournals.org/ by guest on March 5, 2014
847
Mechanisms Responsible for the
Heterogeneous Coronary Microvascular
Response to Nitroglycerin
Michael A. Kurz, Kathryn G. Lamping, James N. Bates,
Charles L. Eastham, Melvin L. Marcus,t and David G. Harrison
Nitroglycerin dilates large (.100 ,um) but not small coronary arterial microvessels, and a
putative metabolite of nitroglycerin, S-nitroso-L-cysteine, has been shown in vitro to dilate both
large and small coronary microvessels. Based on this evidence, we tested the hypothesis that the
lack of response of small coronary microvessels was due to an inability of small coronary
microvessels to convert nitroglycerin into its vasoactive metabolite and examined possible
explanations for this phenomenon. We studied left ventricular epicardial microvessels in vivo
using video microscopy and stroboscopic epi-illumination in anesthetized, open-chest dogs.
Diameters were determined while the epicardium was suffused with nitroglycerin, S-nitroso-Lcysteine, or S-nitroso-D-cysteine (all 10 ,M) and nitroglycerin in the presence of L- or
D-cysteine (100 ,uM). None of the agents affected systemic hemodynamics. Nitroglycerin dilated
large arterioles (20±2%) but not small arterioles (1±l1%). Both S-nitroso-L-cysteine and
S-nitroso-D-cysteine were potent dilators of all size classes of microvessels. Concomitant
application of L-cysteine and nitroglycerin evoked dilation in small microvessels (22±4%,
p<0.5 versus nitroglycerin alone) and larger microvessels (27±6%, p=NS versus nitroglycerin
alone). D-Cysteine did not alter the microvascular response to nitroglycerin in either small
(7+4%, p=NS versus nitroglycerin alone) or large (18+3%, p=NS versus nitroglycerin alone)
microvessels. Neither L-cysteine nor D-cysteine had a direct effect on microvascular diameter.
These findings suggest that 1) sulfhydryl groups are required for the conversion of nitroglycerin
to its vasoactive metabolite; 2) the interaction between nitroglycerin and sulfhydryl residues is
a stereospecific process, indicating either an intracellular mechanism or a membraneassociated enzymatic reaction; and 3) a lack of available sulfhydryl groups may be responsible
for the lack of response of small coronary arterioles to nitroglycerin. (Circulation Research
1991;68:847-855)
itroglycerin has been used in the treatment of
acute angina for more than 100 years. Despite its acceptance as a useful agent for
treatment of acute myocardial ischemia, the mechanism by which nitroglycerin exerts its salutary effect
N
From the Department of Internal Medicine and the Cardiovascular Center, College of Medicine, University of Iowa, Iowa City,
Iowa.
Supported by Ischemic Specialized Center of Research grant
HL-32295, National Institutes of Health grant HL-39050, and a
Veterans Administration merit grant. M.A.K. is supported by an
Individual National Research Service Award from the National
Institutes of Health (HL-08093-O1A1). D.G.H. is an Established
Investigator of the American Heart Association.
Address for correspondence: Michael A. Kurz, PhD, Drawer
LL, Cardiovascular Division, Department of Internal Medicine,
Emory University School of Medicine, 1639 Pierce Drive, Atlanta,
GA 30322.
tDr. Melvin L. Marcus died on October 19, 1989.
Received July 10, 1990; accepted November 19, 1990.
only now is becoming well understood. Early studies
by Gorlin et all and later by Winbury et al2,3 showed
that nitroglycerin was a potent dilator of large epicardial coronary arteries but had little or no effect on
total coronary blood flow. These studies suggested
that nitroglycerin had minimal effect on small coronary resistance vessels. Indeed, recent in vivo4 and in
vitro5 studies in our laboratory using direct visualization of coronary microvessels confirmed that nitroglycerin does not dilate small coronary arterioles.
Based on previous reports,5-13 the most plausible
explanation for the heterogeneous microvascular response to nitroglycerin is that nitroglycerin is not
converted to its vasoactive metabolite in small coronary arterioles. Despite extensive investigation, the
mechanism underlying the biotransformation of nitroglycerin to its vasodilator metabolites remains
unclear. The most widely accepted hypothesis holds
that nitroglycerin must interact with a sulfhydryl-
Downloaded from http://circres.ahajournals.org/ by guest on March 5, 2014
848
Circulation Research Vol 68, No 3 March 1991
containing compound to be converted to a vasoactive
intermediate.8,14-21 The most likely candidate for this
sulfhydryl agent is cysteine, because nitroglycerin is
more rapidly converted to active metabolites in the
presence of cysteine than other naturally occurring
thiols.16,20 This interaction probably results in the
production of a nitrosothiol, such as S-nitrosocysteine.13,1617 It is not clear whether this nitrosothiol
directly activates guanylate cyclase or must be further
reduced to release free nitric oxide (NO), which then
activates guanylate cyclase. In the present study, we
sought to determine whether a putative metabolite of
nitroglycerin, S-nitroso-L-cysteine, could dilate small
coronary microvessels in vivo, as it was previously
shown to do in vitro.5 We then sought to determine
whether small coronary microvessels could convert
nitroglycerin to its vasoactive metabolites if supplemented with cysteine. To accomplish these objectives, we used an in vivo preparation to study coronary microvascular responses to S-nitroso-L-cysteine
and to concurrent application of L-cysteine and nitroglycerin. To determine whether the interaction
between nitroglycerin and cysteine was a stereospecific process (i.e., required activity of enzyme), studies also were performed using the nonphysiological
enantiomers S-nitroso-D-cysteine and D-cysteine.
Materials and Methods
General Preparation
Adult mongrel dogs (3-11 kg) of either sex were
anesthetized with ketamine (25 mg/kg i.m.) and
acepromazine (0.2 mg/kg i.m.) followed by a-chloralose (80 mg/kg i.v.). Cannulas were introduced into
both femoral arteries and advanced into the aorta for
the monitoring of aortic pressure, for withdrawal of
blood samples for determination of arterial blood
gases and pH, and for withdrawal of reference blood
flow samples for measurement of myocardial perfusion. A femoral vein was cannulated for administration of drugs and fluids. The chest was opened in the
left fifth intercostal space, and the heart was suspended in a pericardial cradle. A solid-state pressure
transducer (Millar Instruments, Houston) was inserted into the left ventricle via the left atrial appendage and was used to measure left ventricular pressure. The left atrium also was cannulated for
injection of the radiolabeled microspheres used to
determine myocardial perfusion. The epicardial surface of the heart was bathed continuously with
Krebs-Hensleit bicarbonate buffer (37°C, bubbled
with 20% 02-5% C02, balance N2) to maintain the
physiological environment of the epicardium. Body
temperature was maintained at 37°C with a servocontrolled thermal blanket.
High-Frequency Ventilation
Respiratory-induced motion of the heart was minimized by using high-frequency jet ventilation synchronized to the cardiac cycle. After tracheal intubation with a cuffed endotracheal tube, a catheter was
passed through the lumen of the endotracheal tube
and advanced to the pulmonary carina. Compressed
air (5-15 psi) was injected through the catheter for
25-90 msec by a solenoid valve triggered by the left
ventricular dP/dt signal. The catheter was small
enough to allow expired gases to freely pass out of
the endotracheal tube. Positive end-expiratory pressure was provided by placing the end of the expiration tube under 1-3 cm water. Arterial blood gases
and pH were maintained within the physiological
range by varying the duration of inflation, the pressure of the air injected, the position of the air
injection catheter relative to the pulmonary carina,
and the end-expiratory pressure.
Microscope and Illumination System
The heart was positioned under a vertically
mounted incident illumination system (Carl Zeiss,
Inc., Thornwood, N.Y.). The table beneath the animal
could be moved in an x-y plane to allow visualization
of multiple vascular fields in a single heart. The
epicardial surface was epi-illuminated at a single point
of each cardiac cycle with a xenon arc stroboscopic
light source (Chadwick Helmuth, El Monte, Calif.)
and the Zeiss incident illumination system. Zeiss x 6.3
and x 10 (0.20 and 0.25 numerical aperatures) objectives were used with a x 10 eyepiece. A PDP 11/73
computer (Digital Equipment Corp., Chelmsford,
Mass.) controlled the triggering of the strobe (15-25msec duration) using the first positive slope of the left
ventricular dP/dt signal as the initial trigger point. The
delay between the initial trigger point and the strobe
flash was adjusted so that the microvasculature was
visible only at a single point during mid-to-late diastole. Microvascular arteries and veins were differentiated by observing the sequence of illumination of
vessels after a left atrial injection of fluoresceinlabeled dextran (fluorescein isothiocyanate-dextran,
497,000 Da [Sigma Chemical Co., St. Louis], 50 mg/ml
in 0.9% saline). With this technique, arterioles fluoresced first, followed by venules.
Measurements of Microvascular Diameters
Images of microvessels were obtained with a silicone-intensified tube video camera (Dage-MTI, Inc.,
Michigan City, Ind.) and a video digitizer (Image
Technology, Woburn, Mass.). The video camera was
optically coupled to the intravital microscope. Images
were digitized and displayed on a high-resolution
monitor (Panasonic, Secaucus, N.J.). Selected images
during an intervention were stored in the computer
for later analysis using a digitizing tablet (Summagraphics Corp., Cambridge, Mass.). Microvascular
diameters were measured as the average distance
between two lines drawn on the luminal edges of the
vessel wall. The monitor screen was calibrated by
projecting a micrometer grid in the microscope field
and by determining the ratio of micrometer length to
video length. The pixel size of the digitized images
was 4 ttm with a x 6.3 objective and 2 gm with a x 10
objective. We have shown previously that the spatial
Downloaded from http://circres.ahajournals.org/ by guest on March 5, 2014
Kurz et al Heterogeneous Microvascular Responses to Nitroglycerin
resolution of the digitized images is 5 ,um for the x 10
objective and that the interobserver variability in the
measurement of arteriolar diameters is negligible
using this technique.22
Measurement of Myocardial Perfusion
Measurements of myocardial perfusion (radioactive microsphere technique) were used to establish
that the experimental preparation was within the
normal physiological range and remained stable
throughout each procedure. Because drugs were
applied topically, changes in myocardial perfusion
likely occurred only very near the epicardial surface,
and because the microsphere technique may not be
accurate with very small tissue samples, no attempt
was made to determine changes in myocardial flow
evoked by these vasoactive agents. Microspheres
diameter) labeled with 46Sc, 85Sr,
(lx106,
or 114in were
1l3Sn1n 141Ce,15-gm
103Ru, 51Cr,and57co,95 19Cd
injected into the left atrial
vortexed (5 minutes)
appendage, followed by a saline flush (2 ml). Before
and 90 seconds after microsphere injection, a reference blood flow sample was withdrawn at a constant
rate from the femoral artery. Tissue and reference
blood flow samples were counted in a germanium
crystal gamma counter (Canberra Industries, Inc.,
Meriden, Conn.), and myocardial blood flow (MBF)
was calculated using the equation MBF (ml/minx 100
g)=[CMxW]/CR, where CM is sample counts
(counts per time per gram), W is withdrawal rate of
reference sample (milliliters per minute), and CR is
reference blood flow counts (counts per time).
Preparation of Drugs
Our method for synthesizing S-nitroso-L-cysteine
has been described previously.23 Briefly, L-cysteine
(1 mmol HCl salt, Sigma) was dissolved in 1 ml
methanol to produce a 1 M solution. Twenty-five
milliliters of oxygen and 50 ml NO were mixed in a
gas-tight syringe at room temperature to form NO2
gas. This gaseous mixture was injected into a vacutainer tube in dry ice until frozen (several seconds).
The frozen NO2 then was dissolved into the cysteine
methanol solution in a vial surrounded by dry ice to
produce a 1 M stock solution of S-nitroso-L-cysteine. The purity of the S-nitroso-L-cysteine solutions prepared in this manner has been previously
verified with high-performance liquid chromatography. The stock solution was stored at -20°C in the
absence of light. Immediately before application,
S-nitroso-L-cysteine stock solutions were diluted to
10 times the dose to be applied in deoxygenated,
ultrapure water at room temperature. This solution
then was infused into the warmed, oxygenated
Krebs' suffusate and dripped onto the epicardial
surface over the vessels being observed. The rate of
superfusion was calibrated so that the S-nitroso-Lcysteine solution was diluted by a factor of 10,
warmed, oxygenated, and delivered to the epicardial surface in Krebs' buffer at a rate of 2 ml/min.
S-Nitroso-D-cysteine was prepared using an identi-
849
cal technique, except that D-cysteine was substituted for L-cysteine. L-Cysteine and D-cysteine stock
solutions were prepared each day by dissolving in
ultrapure water to a 1 mM concentration.
To determine whether the physiological half-life of
S-nitroso-D-cysteine was identical to that of S-nitroso-L-cysteine, left circumflex coronary artery rings
were studied using a superfusion bioassay preparation. Vessels were preconstricted with prostaglandin
F2a, and relaxations to either S-nitroso-D-cysteine or
S-nitroso-L-cysteine were examined. The transit time
of the nitrosothiols from infusion into oxygenated
Krebs' buffer to the vascular ring was varied (12, 27,
or 40 seconds). Using this technique, the physiological half-life of S-nitroso-L-cysteine in oxygenated
aqueous solution was found to be 34±13 seconds,
which is similar to the half-life described by previous
investigators.24 The physiological half-life of S-nitroso-D-cysteine was identical to that of the L-isomer
(35±9 seconds, p=NS versus S-nitroso-L-cysteine).
To be certain that the rates of oxidation of L- and
D-cysteine were the same in our preparation, additional experiments were performed with Ellman's
reagent (5,5'-dithiobis-[2-nitrobenzoic acid]),25 a colorimetric test specific for reduced thiol compounds.
These studies indicated that the compounds were
identical when prepared and the rates of oxidation
were identical over 5 hours.
Experimental Protocols
To examine the effects of nitroglycerin on microvascular diameters, control diameters and myocardial
perfusion were measured, and nitroglycerin (10 ,M
in warmed Krebs' buffer) was suffused over the
epicardial surface directly under the microscope objective. Microvascular diameters then were recorded
at steady state (after 15-20 minutes of nitroglycerin
superfusion). Local superfusion of nitroglycerin at
this dose did not affect systemic arterial blood pressure or heart rate. After vessel responses were recorded, nitroglycerin suffusion was discontinued, and
30-60 minutes was allowed for vessels to return to
control diameters. Diameters and myocardial perfusion again were determined to establish that the
preparation had remained stable. Microvascular responses to nitroglycerin were studied in 30 vessels in
10 dogs (one to four vessels per dog). Control vessel
diameters ranged from 29 to 267 ,um.
To determine whether a putative metabolite of
nitroglycerin could dilate coronary microvessels in
vivo, responses to S-nitroso-L-cysteine were measured
in 13 dogs (36 vessels, one to four vessels per dog;
control diameters, 26-300 gm). After control diameters and myocardial perfusion were recorded, S-nitroso-L-cysteine was added to the Krebs' superfusate
to provide 2 ml/min of 1 nM to 100 ,M S-nitroso-Lcysteine. To minimize decomposition of the S-nitrosoL-cysteine before it reached the epicardial surface,
S-nitroso-L-cysteine was added to the warmed, oxygenated Krebs' so that exposure to oxygen was limited
to approximately 1.5 seconds. Responses were deter-
Downloaded from http://circres.ahajournals.org/ by guest on March 5, 2014
850
Circulation Research Vol 68, No 3 March 1991
mined after 15-20 minutes of S-nitroso-L-cysteine
suffusion. In eight dogs, S-nitroso-L-cysteine suffusion
was stopped for 30-60 minutes to allow vessels to
return to baseline diameter before repeating applications of S-nitroso-L-cysteine. In these dogs, S-nitrosoL-cysteine was applied in randomly selected doses (1
nM-100 ,uM), with control diameters recorded before
and after each application. In five other dogs, S-nitroso-L-cysteine was applied in increasing concentrations without control diameters recorded between
applications. Final control diameters and myocardial
blood flows were determined in these experiments to
establish that the preparation had remained stable.
No more than three applications of S-nitroso-L-cysteine were performed in any one experiment to minimize any potential for development of tolerance or
tachyphylaxis.
To determine whether response to S-nitroso-Lcysteine might be attributable to extracellular mechanisms, responses to S-nitroso-D-cysteine also were
measured (16 vessels in four dogs). Because S-nitroso-D-cysteine is a highly polar molecule (and thus
would not diffuse freely across the sarcolemma) and
sarcolemmal transport mechanisms are generally stereospecific for levorotary isomers, it would not be
expected to cross the sarcolemma. Because 10 ,M
S-nitroso-L-cysteine caused a maximum dilatory response in all size classes of coronary microvessels,
this concentration was used to determine microvascular responses to S-nitroso-D-cysteine. The protocol
for applying S-nitroso-D-cysteine was the same as
that used for application of S-nitroso-L-cysteine.
To determine the role of thiol groups in the
heterogeneous response to nitroglycerin, nitroglycerin was applied to the epicardial surface of six dogs
after 30 minutes of L-cysteine suffusion (n=23 vessels). Control diameters and flow were recorded, and
L-cysteine was added to the superfusate to produce
an epicardial suffusion of 100 1uM L-cysteine at 2
ml/min. After 30 minutes, control diameters again
were recorded, and nitroglycerin was added to the
L-cysteine-Krebs' suffusate. Response diameters
were recorded after 15-20 minutes. The nitroglycerin-L-cysteine superfusion then was stopped, and final
control diameters and myocardial flows were recorded. This same protocol was performed with
D-cysteine substituted for L-cysteine (22 vessels in six
dogs) to determine whether vasoactive responses to
cysteine-nitroglycerin coinfusion could be attributed
to extracellular, nonstereospecific mechanisms.
Criteria for an Acceptable Experiment
Experiments were not conducted in animals in
which mean arterial blood pressure was less than 60
mm Hg or if arterial blood gases or myocardial
perfusion were out of the normal physiological
ranges (pH 7.35-7.45, Pco2 25-40 mm Hg, Po2>70
mm Hg, and transmural flow <200 ml/minx 100 g).
In addition, experiments were excluded from the
study if mean arterial blood pressure or heart rate
changed by more than 10% during the protocol
(n =4) or if vessel diameters did not return to within
10% of the original control diameters after application of drugs (n=7).
Statistical Analysis
All data are expressed as mean±SEM. For evaluation of responses to topical application of nitroglycerin and nitrosothiols, response diameters were compared with the control diameter recorded immediately
before the application of the compound. Responses
evoked by application of nitroglycerin and L- or D-CYSteine were compared with the diameter recorded after
a 30-minute preincubation with cysteine and immediately before nitroglycerin was added to the superfusate.
Because responses to nitroglycerin differed markedly
depending on vessel size, microvessels were divided
into two groups (control diameter <100 ,um and >100
,uM) for analysis. These size classes also were used for
comparison of heterogeneous responses to all other
drugs studied. Student's paired t test was used in
comparing response diameters to control diameters.26
Analysis of variance and the Newman-Keuls multiple
comparison procedure were used to compare responses
between groups.26 Responses were considered significant atp<0.05.
Results
Systemic arterial blood pressure and heart rate
were within the normal range for all groups and were
not affected by topical application of any of the
agents studied (Table 1). Transmural myocardial
perfusion also was within the normal range and did
not change during the course of the study (beginning,
114±8 ml/minx 100 g; termination, 110±9 ml/
minx 100 g, p=NS). Responses evoked by epicardial
application of nitroglycerin (10 ,uM, 2 ml/min) varied
with vessel size (Figure 1). Small vessels (control
diameter< 100 ,um) did not dilate (1 ± 1%), but large
vessels dilated 20+2% (p<0.05 versus small vessels).
The maximum dilatory responses to S-nitroso-Lcysteine occurred at a superfusion concentration of
10 ,uM (Figure 2). S-Nitroso-L-cysteine dilated both
small and large epicardial coronary arterioles
(39±7% and 19±2%, respectively, Figure 3). S-Nitroso-D-cysteine (10 ,M) elicited similar responses in
small and large vessels (+32±5% and 23±4%, respectively, Figure 4). These responses were comparable to those evoked by S-nitroso-L-cysteine.
Incubation with either L-cysteine or D-cysteine
alone (100 KM) had no direct effect on large or small
microvessels (Table 2). Coronary microvascular responses to nitroglycerin after preincubation with
L-cysteine or D-cysteine are shown in Figure 5. D-Cysteine did not alter the response to nitroglycerin in
either small or large coronary arteries (+7±4% and
+18+±3%, respectively, p=NS versus nitroglycerin
alone). L-Cysteine also did not change the largevessel response to nitroglycerin (+27±6%, p=NS
versus nitroglycerin alone), but after preincubation
with L-cysteine, nitroglycerin produced dilation in
small coronary arteries comparable to that of large
Downloaded from http://circres.ahajournals.org/ by guest on March 5, 2014
Kurz et al Heterogeneous Microvascular Responses to Nitroglycerin
851
TABLE 1. Systemic Hemodynamic Responses to Topical Application of Various Treatments to Left
Ventricular Epicardium
MABP
Treatment
100+3
104±5
88±4
97±4
94±6
Control
99+3
105±5
87±5
96±4
96±5
86±7
88±7
Treatment
p
NS
NS
NS
NS
NS
NS
NS
Heart rate
Treatment
132+6
141±7
151±14
133±9
141±8
155±5
155 ±5
Control
135+7
140±5
153±15
128±7
135±11
155±5
155 ±5
p
NS
NS
NS
NS
NS
NS
NS
GTN (n=10)
L-cysNO (n=13)
D-cysNO (n=4)
L-cys (n=8)
L-cys+GTN (n=6)
89±8
D-cys (n=6)
87±7
D-cys+GTN (n=6)
Values are mean±SEM. MABP, mean arterial blood pressure; GTN, nitroglycerin; L-cysNO, S-nitroso-L-cysteine;
D-cysNO, S-nitroso-D-cysteine; L-cys, L-cysteine; D-cys, D-cysteine.
vessels (+22+4%,p =NS versus large vessels,p<0.05
versus nitroglycerin alone).
Data are summarized and expressed as percent
change from control diameter in Figure 6. Analysis of
variance and multiple comparison procedures revealed that the data fell into two groups. In one
group, responses were similar to the response evoked
by nitroglycerin in small microvessels (i.e., no effect).
Included in this group are large- and small-microvessel responses to L-cysteine and D-cysteine alone, as
well as small-vessel responses to D-cysteine plus
nitroglycerin. The second group is composed of
small- and large-microvessel responses to S-nitrosoL-cysteine, S-nitroso-D-cysteine, and L-cysteine plus
nitroglycerin, as well as large-microvessel responses
to D-cysteine plus nitroglycerin. Responses to these
agents were found to be similar to the response
evoked by nitroglycerin in large coronary arterioles.
Discussion
The present experiments provide important new
information regarding the biotransformation of nitroglycerin to its active vasodilator metabolite. By
studying vessels in vivo that do not normally relax in
response to nitroglycerin, we have tried to clarify the
350
biochemical intermediates in this pathway. We have
further tried to provide insight into why nitroglycerin
does not dilate small coronary microvessels. Our
findings clearly show that coronary arterioles with
diameters less than approximately 100 gm do not
dilate in response to nitroglycerin, thus confirming
the reports of previous investigators.45 We also demonstrated for the first time that a putative metabolite
of nitroglycerin, S-nitroso-L-cysteine, could relax
small coronary resistance vessels in vivo, and we were
able to show that nitroglycerin evokes relaxation in
small coronary microvessels in the presence of exogenous L-cysteine.
Mechanism of Nitroglycerin-Induced Dilation
NO, or an NO-containing compound, generally has
been accepted as the active vasodilator metabolite of
nitroglycerin, but the mechanism by which nitroglycerin is metabolized to NO has not been determined
45
r
A
O0
CONTROL Dc 100 pm
,10 WOOm
CONTROL D
cc 30
W
r
0
E
z
=L
15
CM
100
oL
0
CONTROL
GTH
FIGURE 1. Coronary arteriolar responses to 10 pMnitroglycerin (GTN). Large symbols represent means for groups with
control diameters less than 100 ,an (open circles) and control
diameters greater than 100 tam (filled circles) ±SEM. Nitroglycerin dilated large vessels from 183±12 to 218 +15 ,um but
had no effect on small vessels (58.5+5 to 58.9+5 pm).
*Different from control value, p <0. 05.
-9
-8
-7
-6
-5
-4
log [L-cysNO]
FIGURE 2. Dose-response curve for S-nitroso-L-cysteine
(L-cysNO). Significant dilation occurred with 1 pML-cysNO.
Maximum response in large vessels occurred with 1 ttM
L-cysNO; maximum response in small vessels occurred at 10
pM. *Significant response, p<O.05. tDifferent from largevessel response, p <0. 05. D, diameter.
Downloaded from http://circres.ahajournals.org/ by guest on March 5, 2014
852
Circulation Research Vol 68, No 3 March 1991
300
250 r
r
0
0~~~~~~~
E
200
W0
=L
~~~~~0
0
Ul
=L
c-
W
~~~~0
OD
a
150
0
100 .
*
50
L
CYSTEINE
0
CONTROL
L-cysNO
FIGURE 3. Changes in coronaty microvascular diameter
evoked by 10 pM S-nitroso-L-cysteine (L-cysNO). Large
vessels dilated from 180±12 to 211±12 pum; small vessels
dilated from 74+6 to 102±9 pum. *Different from control
value, p <0.05. Large symbols represent means for groups with
control diameters less than 100 pum (open circles) and control
diameters greater than 100 pum (filled circles) ±SEM.
precisely. A few recent reports have suggested that
hepatic cytochrome P-450 is capable of denitrating
nitroglycerin and releasing NO.27'28 However, because this reaction is strongly inhibited by oxygen, its
importance in the vasodilatory response to nitroglyc300 [
200~
=L
c-
a
100
Q
0
T.
GOTO
oyN
CYSTEINE +
GTN
FIGURE 5. Effects of L-cysteine (open symbols) and D-cysteine (filled symbols) on the coronary microvascular response
to nitroglycerin (GTN). Group with control diameters greater
than 100 pm is shown with boxes; group with control diameter
less than 100 tun is shown with circles. In the presence of
exogenous L-cysteine, nitroglycenn evoked dilation in small
vessels (61±5 to 75±8 pm, 22±4%o) similar to that evoked in
large vessels (159±13 to 198±14 pm, 27±6%). D-Cysteine
did not alter the response to nitroglycerin in eithergroup (small
vessels: 55±3 to 59±6 pm, 7±4%o, p=NS; large vessels:
147±19 to 174±22 pm, 18±3%). *Significant response,
p < 0. 05. tDifferent from small-vessel response, p < 0. 05.
erin under normal physiological conditions is not
clear.
A majority of the literature suggests that interaction with specific sulfhydryl-containing groups, such
as cysteine, is necessary for nitroglycerin to activate
guanylate cyclase and produce vasodilation. Based on
this literature and the present findings, we suggest
the following mechanism of action for nitroglycerin
(Figure 7). Nitroglycerin interacts with a thiol, probably cysteine, to produce a nitrosothiol (S-nitroso-Lcysteine). Previous investigators have suggested that
this biotransformation involves two separate sulffhydryl-requiring steps.'6,17 It has been suggested that
the first step is an oxidation of cysteine to cystine,
which results in the production of nitrite ion (NO2 ).
NO2 then is protonated to nitrous acid (HONO),
0
FIGURE 4. Changes in coronary microvascular diameter
evoked by 10 MriM S-nitroso-D-cysteine (D-cysNO). Large
vessels dilated from 148+16 to 189±19 pum; small vessels
dilated from 67±6 to 88±9 pum. *Different from control
value, p <0.05. Large symbols represent means for groups with
control diameters less than 100 pm (open circles) and control
diameters greater than 100
(filled circles) ±SEM.
pm
which then interacts with a second sulfhiydryl group
to produce a nitrosothiol.29 This mechanism seems
improbable, however, because NO2 is a weak vasodilator compared with nitroglycerin. If nitroglycerin
had to be converted to NO2 to be activated, then its
potency could not exceed that of NO2J. Furthermore,
the pK of NO2- does not favor the formation of
HONO under physiological conditions. A more likely
TABLE 2. Left Ventricular Epicardial Coronary Microvascular Responses to L-Cysteine or D-Cysteine
Baseline
Thiol
L-cys, D<100 ,m (n=17)
63±5
61+5
185+14
L-cys, D>100 gm (n=15)
175±14
54±4
D-cys, D<100 gm (n=14)
55+3
155+±20
D-cys, D.100 gm (n=8)
Values are mean+SEM. L-cys, L-Cysteine; D-cys, D-cysteine; D, diameter.
147±+19
Downloaded from http://circres.ahajournals.org/ by guest on March 5, 2014
p
NS
NS
NS
NS
Kurz et al Heterogeneous Microvascular Responses to Nitroglycerin
50
r
t
El CTRDc100O±m
E CTR D> 100pmn
lr
t
T
LW
-J
0
25
853
f
U
0
IL
0
FIGURE 6. Summary of responses to nitroglycerin (GTN), S-nitrosocysteine, and cysteine. Responses fall into two groups: those similar to the
small-vessel nitroglycerin response (L-cysteine
[L-CYS], D-cysteine [D-CYS], and small-vessel
D-CYS+GTN); and those similar to the largevessel nitroglycerin response (S-nitroso-L-cysteine
[L-cYsNO], S-nitroso-D-cysteine [D-cYsNO],
L-CYS+GTN, and large-vessel D-CYS+GTN).
*Different from small-vessel response, p<0.05.
tDifferent from large-vessel GTN response,
p<O.OS. CTR D, control diameter.
-10 L
GTN
L-CYS
L-CYS
+ GTN
D-CYS
D-CYS
CYSNO
D-CYSNO
+ GTN
mechanism would involve a direct conversion of
nitroglycerin to a nitrosothiol, probably accomplished by an enzymatic mechanism.
It is not clear whether the initial interaction between nitroglycerin and cysteine occurs inside the
vascular smooth muscle cell or in the extracellular
space. In the present study, S-nitroso-D-cysteine was
found to be as potent a dilator of small microvessels
as S-nitroso-L-cysteine, indicating that nitrosothiol
intermediates can evoke relaxation without entering
the cell, presumably by releasing NO at the cell
membrane. Although this does not directly support
previous evidence of an extracellular pathway for the
interaction of nitroglycerin and cysteine,20,30 it does
suggest that a metabolite of nitroglycerin can evoke
vascular relaxation from the extracellular space. The
most likely mechanism by which NO release would
occur is by a one-electron reduction. This reaction
would result in the release of a thiol ion (SH) and
the NO radical. Because this reduction is not likely to
FIGURE 7. Proposed mechanism of nitroglycerin (GTN) -induced activation ofguanylate cyclase (GC). L-Cysteine (orpossibly
another thiol-containing group, L-R-SH) potentiates the effect of nitroglycerin in small vessels either by interacting with nitroglycerin
at the cell surface to form an S-nitroso-L-thiol (L-R'-S-NO) or by being transported into the cell and interacting with nitroglycerin
there to produce L-RK-S-NO. S-Nitrosothiols containing dextrorotary R'-groups (D-R'-S-NO) such as S-nitroso-D-cysteine also are
potent dilators of all size classes of coronary arterioles, presumably via reduction at the cell membrane and release offree nitric oxide
(NO). D-oriented R-SH groups such as D-cysteine, however, do not interact with nitroglycerin to form vasodilator compounds. For
further description, see "Discussion. "
Downloaded from http://circres.ahajournals.org/ by guest on March 5, 2014
854
Circulation Research Vol 68, No 3 March 1991
occur in the oxygen-rich interstitium, we propose that
an electron is donated by the vascular smooth muscle
sarcolemma to facilitate the reduction (see Figure 7).
Preliminary evidence in our laboratory indicates that
vascular smooth muscle cells possess the capacity to
donate electrons in this manner.
Another significant finding of the present study
was that nitroglycerin relaxed small coronary microvessels in the presence of exogenous L-cysteine,
but not D-cysteine, indicating that a stereospecific
process was involved in the biotransformation. It is
interesting to speculate that the biotransformation of
nitroglycerin requires an enzyme, likely associated
with the plasma membrane, which produces a nitrosothiol from nitroglycerin and cysteine. Indeed, a
very recent report by Chung and Fung31 indicates
that the biotransformation of nitroglycerin occurs
primarily in the plasma membrane fragment of vascular smooth muscle cells.
Mechanism Responsible for Heterogeneity of
Nitroglycerin Response
Many investigators have demonstrated that thiols
are required for nitroglycerin to evoke vasodilation.8 13,14-20 The present findings suggest that small
coronary resistance vessels are unresponsive to nitroglycerin because these thiol groups are not available
in sufficient quantity for the biotransformation process. Although it is clearly implausible that thiolcontaining compounds are absent in the smooth
muscle cells of small coronary resistance vessels, it is
conceivable that specific pools of sulfhydryl groups,
which are necessary for the biotransformation of
nitroglycerin and are present in large vessels, are
lacking in very small vessels. Exogenous cysteine had
no effect on larger microvessels in the present study,
possibly because these vessels already possessed a
sufficient supply of available sulfhydryl compounds.
This essential store of sulfhydryl groups would most
likely be associated with the interior of the vascular
smooth muscle cell, because if it were a plasma or
interstitial pool, it would be available to all size
classes of coronary microvessels. The "pool" also
apparently is not located on the outside of the
smooth muscle membrane, because Moffat et a132
have reported that inactivation of membrane-bound
sulfhydryl groups does not affect the response to
nitroglycerin in canine saphenous vein.
The concept that a sulthydryl "pool" exists is new.
Depletion of "available sulfhydryl" groups has long
been implicated in the development of tolerance to
nitroglycerin and other nitrovasodilators.13,14,20,33,34
The preponderance of evidence suggests that nitrate
tolerance can be reduced or reversed by the administration of cysteine either directly or via a "delivery
system" such as N-acetylcysteine20,33,34 or L-2-oxothiazolidine-4-carboxylate,35 although a few recent
studies have presented evidence to the contrary.30,3236 Most of these studies used large-vessel
preparations such as aortic strips or epicardial coronary artery rings, and so their implications for small
arteriole mechanisms are limited. Winniford et a137
showed, however, that intracoronary infusion of
N-acetylcysteine in humans greatly potentiated the
coronary blood flow response to nitroglycerin.
Horowitz et a138 also have shown that N-acetylcysteine potentiates systemic vascular responses to nitroglycerin. These studies strongly suggest that the
addition of cysteine residues potentiates the vasodilatory response to nitroglycerin in small arterial resistance vessels. The present study provides the first
direct in vivo evidence to support this suggestion.
The finding that nitroglycerin is a potent dilator
of small coronary resistance vessels in the presence
of exogenous cysteine may have important clinical
implications. A clinical strategy that has been used
to try to potentiate the vasodilatory effects of
nitrates or reverse nitrate tolerance involves concurrent administration of a sulfhydryl-containing
agent, most often N-acetylcysteine, with nitroglycerin. Our data suggest that the improvement in flow
caused by concurrent administration of cysteine and
nitroglycerin is likely due to dilation of small coronary resistance vessels rather than to an increased
dilation of larger epicardial vessels. This being the
case, the concurrent administration of cysteine with
nitroglycerin might contribute to a "myocardial
steal" phenomenon, rather than actually improving
flow to the ischemic area.
Acknowledgments
The authors wish to thank Jennifer Clothier
and John Clausen for their technical assistance
in the laboratory and Cindy Evans, Maureen
Kent, and Henriette Washington for the preparation of the manuscript.
References
1. Gorlin R, Brachfield N, Macleod C Bopp P: Effect of nitroglycerin on the coronary circulation in patients with coronary
artery disease or increased left ventricular work. Circulation
1959;19:705-718
2. Winbury MM, Howe BB, Hefner MA: Effect of nitrates and
other coronary dilators on large and small coronary vessels:
An hypothesis for the mechanism of action of nitrates. J
Pharmacol Exp Ther 1969;168:70-95
3. Winbury MM, Howe BB, Weiss HR: Effect of nitroglycerin
and dipyridamole on epicardial and endocardial oxygen tension: Further evidence for redistribution of myocardial blood
flow. J Pharmacol Exp Ther 1971;176:184-199
4. Kanatsuka H, Lamping K, Eastham C Marcus M: Nonuniform effect of nitroglycerin on different size coronary
arterial microvessels (abstract). FASEB J 1988;2:A496
5. Sellke FW, Meyers PR, Bates JN, Harrison DG: Influence of
vessel size on the sensitivity of porcine coronary microvessels
to nitroglycerin. Am J Physiol 1990;258:H515-H520
6. Katsuki S, Arnold W, Mittal C, Murad F: Stimulation of
guanylate cyclase by sodium nitroprusside, nitroglycerin and
nitric oxide in various tissue preparations and comparison to
the effects of sodium azide and hydroxylamine. J Cyclic
Nucleotide Res 1977;3:23-35
7. Kukovetz WR, Holzmann S, Wurm A, Poch G: Evidence for
cyclic GMP-mediated relaxant effect of nitro-compounds in
coronary smooth muscle. Naunyn SchmiedebergsArch Pharma-
col 1979;350:129-138
8. Gruetter DY, Gruetter CA, Barry BK, Baricos WH, Hyman
AL, Kadowitz PH, Ignarro LJ: Activation of coronary arterial
Downloaded from http://circres.ahajournals.org/ by guest on March 5, 2014
Kurz et al Heterogeneous Microvascular Responses to Nitroglycerin
9.
10.
11.
12.
13.
guanylate cyclase by nitric oxide, nitroprusside, and nitrosoguanidine-inhibition by calcium, lanthanum and other cations
enhancement by thiols. Biochem Pharmacol 1980;29:
2943-2950
Rapoport RM, Draznin MB, Murad F: Endothelium-dependent
relaxation in rat aorta may be mediated through cyclic GMP-dependent protein phosphorylation. Nature 1983;306:174-176
Ignarro LJ, Harbison RG, Wood KS, Kadowitz PJ: Activation
of purified soluble guanylate cyclase by endothelium-derived
relaxing factor intrapulmonary artery and vein: Stimulation by
acetylcholine, bradykinin and arachidonic acid. J Pharnacol
Exp Ther 1986;237:893-900
Rapoport RM, Waldman SA, Ginsburg R, Molina CR, Murad
F: Effects of glyceryl trinitrate on endothelium-dependent and
-independent relaxation and cyclic GMP levels in rat aorta and
human coronary artery. J Cardiovasc Pharmacol 1987;10:82-89
Tsai S, Adamik R, Monganiello VC, Moss J: Effects of
nitroprusside and nitroglycerin on cGMP content and PGI2
formation in aorta and vena cava. Biochem Pharmacol 1989;
38:61-65
Romanin C, Kukovetz WR: Tolerance to nitroglycerin is
caused by reduced guanylate cyclase activation. J Mol Cell
Cardiol 1989;21:41-48
14. Needleman P, Johnson EM Jr: Mechanism of tolerance development to organic nitrates. J Pharmacol Exp Ther 1973;184:
709-715
15. Needleman P, Jakschik B, Johnson EM Jr: Sulfhydryl require16.
17.
18.
19.
20.
21.
22.
ment for relaxation of vascular smooth muscle. J Pharmacol
Exp Ther 1973;187:324-331
Ignarro LJ, Gruetter CA: Requirement of thiols for activation
of coronary arterial guanylate cyclase by glyceryl trinitrate and
sodium nitrite. Biochim Biophys Acta 1980;631:221-231
Ignarro LJ, Lippton H, Edwards JC, Baricos WH, Hyman AL,
Kadowitz PJ, Gruetter CA: Mechanism of vascular smooth
muscle relaxation by organic nitrates, nitrites, nitroprusside
and nitric oxide: Evidence for the involvement of S-nitrosothiols as active intermediates. J Pharmacol Etp Ther 1981;218:
739-749
Feelisch M, Noack E: Nitric oxide (NO) formation from
nitrovasodilators occurs independently of hemoglobin or nonheme iron. Eur J Pharmacol 1987;142:465 -469
Rapoport RM, Murad F: Effects of ethacrynic acid and
cystamine on sodium nitroprusside induced relaxation, cyclic
GMP levels and guanylate cyclase activity in rat aorta. Gen
Pharmacol 1988;19:61-65
Fung HL, Chong S, Kowaluk E, Hough K, Kakemi M:
Mechanisms for the pharmacologic interaction of organic
nitrates with thiols: Existence of an extracellular pathway for
the reversal of nitrate vascular tolerance by N-acetylcysteine. J
Pharmacol Exp Ther 1988;245:524-530
Schroder H, Noack E, Muller R: Evidence of a correlation
between nitric oxide formation by cleavage of organic nitrates
and activation of guanylate cyclase. J Mol Cell Cardiol 1985;
17:931-934
Lamping KG, Kanatsuka H, Eastham CL, Children WM,
Marcus ML: Non-uniform vasomotor responses of the coro-
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
855
nary microcirculation to serotonin and vasopressin. Circ Res
1989;65:343-351
Bates JN, Myers PR, Guerra R, Baker MT, Harrison DG:
Synthesis and detection of S-nitroso-L-cysteine (abstract).
FASEB J 1989;3:A1144
Myers PR, Minor RL Jr, Guerra R Jr, Bates JN, Harrison DG:
Vasorelaxant properties of the endothelium-derived relaxing
factor more closely resemble S-nitrosocysteine than nitric
oxide. Nature 1990;345:161-163
Ellman GL: Tissue sulfhydryl groups. Arch Biochem Biophys
1959;82:70-77
Rosner BA: Fundamentals of Biostatistics. Boston, Duxbury,
1982
Servents D, Delaforge M, Ducrocq C, Mansuy D, Lenfant M:
Nitric oxide formation during microsomal hepatic denitration
of glyceryl trinitrate: Involvement of cytochrome P-450. Biochem Biophys Res Commun 1989;163:1210-1216
McDonald BJ, Bennett BM: Biotransformation of organic
nitrates by rat hepatic cytochrome P-450 (abstract). FASEB J
1990;4:1099
Romanin C, Kukovetz WR: Guanylate cyclase activation by
organic nitrates is not mediated via nitrite. J Mol Cell Cardiol
1988;20:389-396
Munzel T, Holtz J, Mulsch A, Steward DJ, Bassenge E: Nitrite
tolerance in epicardial arteries or in the venous system is not
reversed by N-acetylcysteine in vivo, but tolerance-independent
interactions exist. Circulation 1989;79:188-197
Chung SJ, Fung HL: Identification of the subcellular site for
nitroglycerin metabolism to nitric oxide in bovine coronary
smooth muscle cells. J Pharmacol Exp Ther 1990;253:614-619
Moffat JA, Armstrong PW, Marks GS: Investigations into the
role of sulfhydryl groups in the mechanism of action of the
nitrates. Can J Physiol Pharmacol 1982;60:1261-1266
May DC, Popma JJ, Black WH, Schaefer S, Lee HR, Levine
BD, Hillis LD: In vivo induction and reversal of nitroglycerin
tolerance in human coronary arteries. N Engl J Med 1987;317:
805-809
34. Packer M, Lee WH, Kessler PD, Gottlieb SS, Medina N,
Yushak M: Prevention and reversal of nitrate tolerance in
patients with congestive heart failure. N Engl J Med 1987;317:
799-804
35. Kukovetz WR, Holemann S, Mayer B: Prevention of tolerance
to organic nitrates by an intracellular cysteine delivery system
(OTC), in Fujiwara M, Narumiya S, Miwa S (eds): Biosignaling
in Cardiac and Vascular Systems. Elmsford, NY, Pergamon
Press, Inc, 1989, pp 403-409
36. Gruetter CA, Lemke SM: Dissociation of cysteine and glutathione levels from nitroglycerin-induced relaxation. Eur J
Pharmacol 1985;111:85-95
37. Winniford MD, Kennedy PL, Wells PJ, Hillis LD: Potentiation of nitroglycerin-induced coronary dilation by N-acetylcys-
teine. Circulation 1986;73:138-142
38. Horowitz JD, Aatman EM, Lorell BH, Barry WH, Smith TW:
Potentiation of the cardiovascular effects of nitroglycerin by
N-acetylcysteine. Circulation 1983;68:1247-1256
KEY WoRDs
* cysteine
nitroglycerin * coronary arteries * microcirculation
S-nitrosocysteine
Downloaded from http://circres.ahajournals.org/ by guest on March 5, 2014