© 1996 American Heart Association, Inc.
Articles |
Low Increase in cGMP Induced by Organic Nitrates and Nitrovasodilators Improves Contractile Response of Rat Ventricular Myocytes
From the Institut für Pharmakologie (G.K., K.K., P.N., E.N.), Heinrich-Heine-Universität, Düsseldorf, Germany, and the Institut für Physiologie (K.D.S., H.M.P.), Justus-Liebig-Universität, Gießen, Germany.
Correspondence to Dr Georg Kojda, Institut für Pharmakologie, Heinrich-Heine-Universität, Moorenstr 5, 40225 Düsseldorf, Germany.
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Abstract |
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 Top Abstract IntroductionMaterials and Methods Results Discussion References |
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Abstract Whether organic nitrates are bioactivated to NO in cardiac muscle cells and may thus directly affect cardiac contractile function has remained an open question. Therefore, we determined the effects of the organic nitrates glyceryl trinitrate (100 µmol/L), pentaerythritol tetranitrate (10 µmol/L), and isosorbide-5-mononitrate on electrically stimulated contractile response (CR) and cAMP and cGMP content of isolated adult rat ventricular cardiomyocytes compared with different concentrations of the spontaneous NO donors S-nitroso-N-acetyl-d,l-penicillamine (SNAP) and 2,2-diethyl-1-hydroxy-1-nitroso-hydrazine (DEA/NO). A high concentration of spontaneous NO donors (100 µmol/L) caused a large increase in cGMP content that was accompanied by a decrease in CR to 73.8±6.7% (SNAP) and 80.9±6.1% (DEA/NO) of the control values. Inhibition of cGMP-dependent protein kinase by 10 µmol/L KT 5822 converted this effect into a pronounced improvement of CR (163.5±14.0%). By contrast, the organic nitrates caused a small but significant increase in cGMP, which was accompanied by an increase in cAMP and CR identical to that induced by 10 nmol/L isoprenaline (141.6±6.4%). A similar effect was observed with a low concentration (1 µmol/L) of SNAP and DEA/NO. All increases in CR induced by nitrates were abolished after inhibition of cAMP-dependent protein kinase by Rp-cAMPS (10 µmol/L). The positive contractile effect of isoprenaline was enhanced by 1 µmol/L SNAP. This effect was also demonstrated in isolated rat papillary muscles. These results indicate that in cardiac muscle (1) organic nitrates are bioactivated to NO; (2) this results in a moderate increase in cGMP, which causes an improved CR by increasing cAMP and activating cAMP-dependent protein kinase; and (3) a large increase in cGMP, produced by high doses of NO donors, reduces CR because of the activation of cGMP-dependent protein kinase.
Key Words: cardiomyocytes • heart muscle • organic nitrates • nitric oxide • contractility
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Introduction |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Organic nitrates and spontaneous NO donors might be considered as two types of the same class of drugs, defined by identical steps of mechanism of action, such as generation of NO, activation of sGC, production of cGMP, and activation of cGMP-dependent protein kinase.1 The main difference between these two types of drugs is in the mechanism of liberation of NO. Organic nitrates like ISMN, GTN, and PETN decompose in vitro solely in the presence of certain thiols2 3 4 and in vivo presumably by the support of enzymatic catalysis.5 6 NO donors like SNAP and DEA/NO spontaneously generate NO without additional cofactors.3 7 Metabolic bioactivation of organic nitrates to NO, demonstrated directly by detection of NO or indirectly by accumulation of cGMP, occurs in different cell types, such as vascular smooth muscle cells, endothelial cells, fibroblasts, or macrophages.6 8 9 In therapy, these drugs are considered to be vasorelaxants acting primarily on the venous side of circulation. The main hemodynamic changes resulting in therapeutic benefit are believed to be preload reduction and coronary vasodilation.
Contrary to numerous investigations on nitrate action in vascular smooth muscle, effects of these drugs in heart muscle have rarely been studied.1 It has been shown that cGMP, the second messenger generated after stimulation of sGC, reduces calcium influx at isolated ventricular cardiomyocytes.10 According to more recent studies, this action is mediated by cGMP-dependent protein kinase.11 Elevation of cGMP in rat ventricular myocytes has been shown to elicit depressive actions on the beating rate.12 Accordingly, sodium nitroprusside inhibits their contractile response.13 Inhibition of contractile response by high concentrations of NO has also been demonstrated in isolated papillary muscles.14 Under certain conditions, cGMP may also enhance the action of isoprenaline on cardiac calcium influx.15 Recent investigations with rabbit working heart preparations suggest that not only spontaneous NO donors but also organic nitrates influence heart muscle directly.16 In the cited study, ISMN and PETN markedly enhanced the effect of norepinephrine on cardiac output and dP/dtmax. To explain the effects observed and to comparatively investigate actions of organic nitrates and spontaneous NO donors on heart muscle, we measured the influence of these drugs on the basal concentration of cGMP and on the contractile response of rat ventricular myocytes. We found that both types of drugs increase basal levels of cGMP and cAMP. A moderate increase in basal cGMP was associated with an improved contractile response, whereas marked elevations of cGMP decreased contractile activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Preparation of Rat Ventricular Myocytes
Ventricular myocytes were isolated from male Wistar rats (200 to 250 g) as described in detail previously.17 18 Hearts were rapidly excised in deep ether anesthesia, rapidly transferred to ice-cold saline, and mounted on the cannula of a Langendorff perfusion system. Heart perfusion and subsequent steps were all performed at 37°C. First, hearts were perfused in a nonrecirculating manner for 5 minutes at 10 mL/min (composition of perfusate [mmol/L]: NaCl 110, KH2PO4 1.2, KCl 2.6, MgSO4 1.2, Na2CO3 25, and glucose 11, 37°C, gassed with 5% CO2/95% O2). Thereafter, perfusion was continued by recirculation of 50 mL of the above perfusate supplemented with 0.06% (wt/vol) crude collagenase and 25 µmol/L CaCl2 at 5 mL/min. After 30 minutes, ventricular tissue was minced and incubated for 20 minutes in recirculating medium with 1% (wt/vol) bovine serum albumin under 5% CO2/95% O2. By gentle pipetting, cells were released from tissue chunks. The resulting cell suspension was filtered through a 200-µm nylon mesh. Twice, the filtered material was washed by centrifugation (3 minutes, 25g) and resuspension in the above perfusate, in which the concentration of CaCl2 was stepwise increased to 0.2 and 0.5 mmol/L. After another centrifugation (3 minutes, 25g), the cells in the pellet were resuspended at 37°C at a density of 1 to 2 mg protein per milliliter in medium 199 supplemented with 4% fetal calf serum, 100 U/mL penicillin, and 100 µg/mL streptomycin. Primary culture of cardiomyocytes was performed by pipetting 1 mL of this cell suspension into 35-mm culture dishes (Falcon 3001) under sterile conditions. After an incubation of those dishes for 3 to 4 hours in an atmosphere of 5% CO2/95% O2 at 37°C, intact cardiomyocytes had adhered at the culture dish and were separated from broken or dead cells by washing the dishes once with culture medium. This preparation of cardiomyocytes is virtually free of nonmyocytes, as demonstrated previously.18 19 Cardiomyocytes can be distinguished from nonmyocytes by their large size and high myosin content. By a procedure previously described,19 it was determined that the cell preparation investigated here contained <0.5% nonmyocytic cells. Preparation and primary culture of cells took 5 hours. After this time, all experiments were started.
Determination of cGMP
Freshly prepared rat ventricular
myocytes that were
adhered to 35-mm culture dishes (Falcon 3001) were washed once with
modified Tyrode's buffer (containing [mmol/L] NaCl 125,
KH2PO4 1.2 , KCl 2.6, MgSO4 1.2,
CaCl2 1, glucose 10, and HEPES 10, pH 7.4) and equilibrated
for 15 minutes in 1 mL of this buffer (37°C). Incubation (37°C) was
started by the addition of 20 µL of an appropriate stock solution of
SNAP, GTN, ISMN, PETN, DEA/NO, or vehicle (for specifications, see
"Substances and Solutions"). The medium was then gently mixed
(some seconds), and experiments were terminated at the indicated time
intervals by acidifying the incubate with 250 µL of ice-cold
HClO4.(50%). The ice-cold acidified cell suspension
was transferred in 1.5-mL plastic tubes (Eppendorf) and
centrifuged at 4°C and 10 000g for 5 minutes. The
pellet was used for protein determination.20 The
supernatant was neutralized (pH 7.4) with
K3PO4, centrifuged again, and
directly used for determination of cGMP by radioimmunoassay using a
125I-labeled antigen. All experiments were performed with
cells of at least three different cell preparations. Changes of basal
cGMP levels are given as percentages related to the basal cGMP level
(vehicle treated) observed in experiments performed in parallel.
The thoracic aortas of some rats used for the preparation of cardiomyocytes were excised and rapidly immersed in cold oxygenated (95% O2/5% CO2) Krebs-Henseleit solution (pH 7.4) of the following composition (mmol/L): Na+ 143.07, K+ 5.87, Ca2+ 1.6, Mg2+ 1.18, Cl- 125.96, HCO3- 25.00, H2PO4- 1.18, SO42- 1.18, and glucose 5.05. Ring segments (5-mm width) were incubated for 3 hours at 37°C in oxygenated Krebs-Henseleit solution, which was changed every 30 minutes. After equilibration, the vessel segments were incubated with GTN or SNAP (100 µmol/L) or vehicle for 2 or 5 minutes, removed from the incubation medium, flash-frozen in liquid nitrogen, and stored at -80°C. Frozen aortic rings were homogenized with a polytron in 1.0 mL ice-cold HClO4 (10%) and then centrifuged at 4500g for 10 minutes. The pellet was used for protein determination20 ; 900 µL supernatant was neutralized (pH 7.4) with K3PO4, centrifuged again, and directly used for determination of cGMP by radioimmunoassay using a 125I-labeled antigen. The recovery rate of protein and cGMP by use of this procedure was determined previously.21
Determination of cAMP
Freshly prepared rat ventricular
myocytes that were
adhered to 35-mm culture dishes (Falcon 3001) were washed once with
modified Tyrode's buffer (composition as described), pH 7.4, and
equilibrated for 15 minutes in 1 mL of this buffer (37°C). Incubation
(37°C) was started by the addition of 20 µL of an appropriate stock
solution of SNAP, GTN, isoprenaline, a combination of SNAP and
isoprenaline, or vehicle. The medium was then gently mixed (some
seconds), and experiments were terminated after 90 seconds by rapid
removal of the buffer and addition of 2 mL of ice-cooled ethanol.
Cells were suspended, and the ice-cold cell suspension was
transferred in 1.5-mL plastic tubes (Eppendorf). Samples were
evaporated to dryness (Speed-vac), reconstituted with buffer, and
centrifuged at 4°C and 10 000g for 5 minutes. The
pellet was used for protein determination.20 The
supernatant was directly used for determination of cAMP by
radioimmunoassay using a tritium-labeled antigen.
Determination of Contractile Activity of Isolated
Cells
The contractile activity was studied by use of a video
microscopic technique as described previously.22 For
experiments, the cells were incubated in a constant volume, ie, 2 mL
per 35-mm dish. The density of cells was low, ie, 105 cells
per 35-mm dish. The pH of the medium remained constant for the duration
of the experiments, even when cells were electrically stimulated.
Briefly, culture dishes containing attached cardiomyocytes
in 2 mL modified Tyrode's buffer were rapidly placed on a
temperature-controlled (37°C) microscopic stage (immediately
after the addition of either 40 µL vehicle or 40 µL of an
appropriate stock solution of isoprenaline, SNAP, GTN, ISMN, PETN,
DEA/NO, or mixtures of isoprenaline with SNAP to adjust the indicated
concentrations). In some experiments, this was preceded by a 5-minute
preincubation period of the cells with either Rp-cAMPS (10
µmol/L) or KT5823 (10 µmol/L) or both, and these compounds were
also present during these experiments. Treatment of rat
cardiomyocytes with a solution of SNAP (100 µmol/L) that
was incubated for 30 minutes at pH 7.4 and 37°C did not affect their
contractile response. This solution did also not activate a
preparation of human platelet sGC to produce cGMP. Two AgCl
electrodes (diameter, 100 µm) fixed on a plastic ring that covered
the culture dish were rapidly inserted to a distance of 1 mm in the
buffer and positioned laterally to leave the microscopic visual field
in their middle free. Application of biphasic electrical stimuli (to
avoid hydrolysis) composed of two equal but opposite rectangular 50-V
stimuli of 0.5-millisecond duration at a frequency of 0.5 Hz could be
started 10 seconds after addition of the nitrates. The contractions of
cardiomyocytes elicited by electrical field stimulation
were stable within the time of investigation. The microscopic picture
in phase contrast was recorded on a tape by using a
charge-coupled-device video camera and a U-matic video
recorder (model VO-5800PS, Sony). The contractions of single cells
were determined from frozen consecutive video frames magnifying the
cell's picture 500-fold on a video monitor screen. Contractions
occurring 15, 90, and 300 seconds after addition of the nitrates (5,
80, and 290 seconds after the onset of cell stimulation) were
analyzed by measuring the slack length 0.5 second before
contraction (A) and the fully contracted cell length (B). Contractile
response (cell shortening) is expressed as percentage:
100x(A-B)/A. Changes in cell shortening induced by any of the
various drugs used in this study were determined by directly comparing
responses of 30 to 40 different cells of one preparation to vehicle
(all ingredients except the drug) and to the drug. Responses of every
single cell in the presence of the drugs are expressed as a percentage
of the mean control response obtained with cells of the same
preparation (100%). Any experiment was repeated with cells from two
other cell preparations. The exact number of vehicle- and
drug-treated individual cells for every drug studied is indicated
in the figure legends.
Determination of Contractile Force of Isolated Rat Left
Ventricular Papillary Muscles
Hearts were rapidly excised from male
Wistar rats (200 to 250 g)
after cervical translocation in deep ether anesthesia and
rapidly transferred to Krebs-Henseleit buffer. Left
ventricular papillary muscles were prepared and mounted in
10-mL water-jacketed organ baths. Inotropic activity was evaluated
on electrically stimulated preparations (4 Hz, 10 milliseconds, 7 V) by
measuring contractile force isometrically by means of a
force-displacement transducer. The muscles were allowed to
stabilize at 37°C for 
45 minutes. Resting tension was 0.5 g.
Application of isoprenaline (0.1 µmol/L) was repeated until the
response of the preparation was reproducible (three or four times).
Then isoprenaline was applied simultaneously with vehicle
(for specification see "Substances and Solutions"), SNAP (1
µmol/L), or DEA/NO (1 µmol/L). In other experiments, SNAP (100
µmol/L) or DEA/NO (100 µmol/L) was applied after a 5-minute
preincubation of the muscles with 10 µmol/L KT5823.
Substances and Solutions
SNAP was synthesized according to
Field et al.23
The reaction product was recrystallized once in methanol.
Analysis of SNAP included thin-layer
chromatography on Merck RP18 plates with methanol/water
at 7:3 (vol/vol) used as a solvent. To determine free sulfide groups,
spray detection was performed with 1.5 g of sodium nitroprusside
dissolved in a 10 mL aliquot of a solution containing 5 mL of 2N
hydrochloric acid, 95 mL methanol, and 10 mL ammonia (25%). We found
no detectable amount of free sulfides. Spray detection of the nitroso
group was achieved by two steps: zinc powder in methanol and sulfanilic
acid/
-naphthylamine at 250 mg each in 30% acetic acid. This
procedure resulted in a concentration-dependent increase in the
colored area. The preparation decomposed at 148°C to 150°C. Its
NO-liberating property was determined by using the oxyhemoglobin
assay.2 With 1 mmol/L of SNAP, the rate of release of NO
was 1.28±0.01 µmol/L per minute (n=3).
ISMN and PETN were generously provided by ISIS-Pharma; GTN (4.404 mmol/L in 154 mmol/L NaCl, directly used as stock solution) was generously provided by Schwarz Pharma AG; DEA/NO24 was a gift of Dr L. Keefer (Frederick, Md); KT5823 was obtained from Calbiochem; Rp-cAMPS was obtained from Research Biochemicals; isoprenaline and crude collagenase were obtained from Serva Feinbiochemika; fetal calf serum and antibiotics were obtained from GIBCO; medium 199 was obtained from Life Technologies; and all other chemicals (analytical grade) were obtained from Merck or Sigma Chemical Co.
Stock solutions of isoprenaline, Rp-cAMPS (10 mmol/L), and ISMN (1 mol/L) in twice-distilled water, of DEA/NO (10 mmol/L) in 10 mmol/L NaOH, and of KT5823, SNAP, and PETN (100 mmol/L) in dimethyl sulfoxide were prepared daily and kept, protected from daylight, on ice until use. All concentrations indicated in the text, figures, and tables are expressed as final bath concentrations.
Statistics
All data were analyzed by one-way ANOVA with a
subsequent Student-Newman-Keuls test (SAS PC Software 6.04, PROC
ANOVA, also used to calculate the plots) and are expressed as
mean±SEM. Significant differences were evaluated by using either
Student's t test or Wilcoxon's test, and a value
of P<.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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Influence of Nitrates on cGMP and cAMP Level in Cardiomyocytes
The average value of basal cGMP was 1.7±0.2 pmol/mg protein and represents the summary of 129 single experiments performed with 34 different preparations. Basal levels of cGMP in the various experiments performed are given in the legends of Figs 1
and
2. Incubation of rat ventricular
myocytes with either SNAP (100 µmol/L) or GTN (100 µmol/L) resulted
in a time-dependent increase in the basal level of cGMP (Fig
1).
This increase was maximal after 1 minute of incubation, was much more
pronounced after using SNAP, and remained stable for at least 5 minutes
in the presence of the drugs (Fig 1). A similar kinetic pattern
was
also observed in rat aortas (Table 1). The increase in
basal level of cGMP induced by different nitrovasodilators was
confirmed by a second series of experiments, in which rat
ventricular myocytes were incubated with these drugs for 2
minutes (Fig 2). A concentration of 440 µmol/L GTN, which
was the
maximal concentration achievable (see "Materials and Methods"),
induced an increase in cGMP similar to that induced at a concentration
of 100 µmol/L GTN. Smaller increases in cGMP were observed after
incubation of the cells with 5 mmol/L ISMN or 10 µmol/L PETN. The
concentrations chosen were related to the maximal vasodilating
concentrations of the drugs as evaluated by preliminary experiments
using either rat aortic or porcine coronary artery rings.
Contrary to the moderate effects of high concentrations of organic
nitrates, a significant influence of SNAP on cGMP production in
cardiomyocytes was observed starting at 1 µmol/L and
increased in a concentration-dependent manner up to 100 µmol/L
(Fig 2). In addition, DEA/NO, another NO donor, exerted a
stimulation
of cGMP production in rat cardiomyocytes that was
similar to that of SNAP with respect to onset, magnitude, and
concentration dependence.
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Nitrates also affected the level of cAMP in
cardiomyocytes.
As shown in Fig 3, incubation with SNAP (1 µmol/L) and
GTN (100 µmol/L) resulted in a significant increase in basal cAMP
that was comparable to the effect of 10 nmol/L isoprenaline. In
addition, a combination of 1 µmol/L SNAP and 10 nmol/L isoprenaline
elicited an increase in cAMP that was significantly greater than the
increase observed after incubation with either of these drugs
alone.
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Influence of Nitrates on Basal Concentration of cGMP in Rat
Aortas
To compare the increase in cGMP induced by nitrovasodilators in
cardiomyocytes and vascular smooth muscle, aortic segments
of the same animals used for the preparation of
cardiomyocytes were incubated for 2 and 5 minutes with GTN
(100 µmol/L) and SNAP (100 µmol/L). In these experiments, basal
values of cGMP were slightly greater than those detected in
cardiomyocytes (Table 1
, Figs 1 and
2). In contrast,
incubation of rat aortas with both nitrovasodilators resulted in a much
more pronounced increase in cGMP (Table 1). As observed in
cardiomyocytes (Fig 1), SNAP was approximately fourfold
more effective than GTN.
Influence of Nitrates on Contractile Response
Cardiomyocytes
were stimulated for contraction by electrical
field stimulation. The mean value of cell shortening in Tyrode's
solution observed in a typical set of control experiments amounted to
10.5±0.2% (n=124) of the resting cell length. This contractile
response was identical in the presence of 10 µmol/L Rp-cAMPS
(10.4±0.6%, n=114) or 10 µmol/L KT5823 (9.1±0.8%,
n=58).
All nitrovasodilators exhibited a significant influence
on the
contractile response of isolated rat cardiomyocytes, as
illustrated in Fig 4. Electrical stimulation of these
cells in the presence of the organic nitrates GTN, PETN, or ISMN
resulted in a significant improvement of the contractile response
compared with electrical stimulation alone (Fig 4A and
4B). The actions
of GTN and PETN exhibited a rapid onset and were nearly maximal after
90 seconds, whereas the action of ISMN was delayed. With the exception
of 440 µmol/L GTN, which showed a less pronounced improvement of
contractile response, the magnitude of the effect of all organic
nitrates was not significantly different after a 5-minute incubation
period (Fig 4A and 4B).
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Contrary to the
data obtained with organic nitrates, spontaneous NO
donors like SNAP or DEA/NO exhibited opposite effects on the
contractile response of electrically stimulated rat
cardiomyocytes (Fig 4C and 4D). At a low
concentration of 1
µmol/L, both SNAP and DEA/NO improved the contractile activity of
these cells. Compared with organic nitrates, this effect occurred
with a similar onset but was less pronounced. By contrast, a
concentration of 100 µmol/L of either NO donor caused a pronounced
attenuation of the contractile response. This effect developed more
slowly and gained statistical significance after 5 minutes (Fig
4C and 4D).
Effect of SNAP in the Presence of KT5823
In order to gain
information about the mechanism of the
depressant activity of NO donors on the contractile response of
electrically stimulated cardiomyocytes, the effect of SNAP
was determined in cells preincubated with 10 µmol/L KT5823, an
inhibitor of cGMP-dependent protein kinase. Such a
pretreatment resulted in a conversion of the depressant activity of 100
µmol/L SNAP, so that a marked improvement of the contractile response
was observed (Fig 5). In addition, this effect occurred
with a similar onset and a slightly more pronounced magnitude compared
with the effect of organic nitrates. An improved contractile response
of cardiomyocytes preincubated with KT5823 was also induced
by a lower concentration of SNAP (10 µmol/L, Fig 5).
Preincubation
with 10 µmol/L KT5823 had no significant influence on the effect of 1
µmol/L SNAP. The contractile response related to the contraction
of control cells (n=92) after 15, 90, and 300 seconds amounted to
106.7± 3.4%, 129±6.8%, and 118±5.9% (n=112),
respectively (data
not significantly different from data obtained with 1 µmol/L SNAP
alone that are shown in Fig 4C).
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Effects of Nitrovasodilators in the Presence of
Rp-cAMPS
In order to elucidate the origin of the stimulatory activity
of nitrovasodilators on the contractile response of electrically
stimulated cardiomyocytes, the effects of these drugs were
determined in the presence of Rp-cAMPS, an inhibitor of
the cAMP-dependent protein kinase. As shown in Fig 6A,
pretreatment of the cells with Rp-cAMPS completely inhibited the
improvement of their contractile response induced by GTN (100
µmol/L). A similar result was obtained for SNAP (1 µmol/L, Fig
6B). Furthermore, Rp-cAMPS also abolished the pronounced
improvement of the contractile response observed after the incubation
of cardiomyocytes with 100 µmol/L SNAP in the presence of
KT5823 (10 µmol/L, Fig 6C).
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Effects of Nitrates on the Activity of Isoprenaline
Isoprenaline strongly improved the contractile response of
cardiomyocytes induced by electrical stimulation, and this
effect occurred in a dose-dependent manner (Fig 7).
Increase in cell shortening rapidly developed during the first 90
seconds of incubation (data not shown). Parallel incubation with 1
µmol/L SNAP significantly enhanced cell response to 10 nmol/L
isoprenaline and induced an improvement of the contractile response
identical to that observed after 30 nmol/L isoprenaline alone (Fig
7).
Also, parallel incubation of 10 nmol/L isoprenaline with a higher dose
of SNAP (10 µmol/L), which itself does not induce a significant
contractile effect (see Fig 4C), significantly enhanced the
contractile
response of cardiomyocytes to isoprenaline but to a lesser
extent (data not shown).
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Effects of Isoprenaline, SNAP, and DEA/NO in Rat Left
Ventricular Papillary Muscle
In isolated rat left ventricular
papillary muscle,
isoprenaline (0.1 µmol/L) increased the development of contractile
force (Table 2). Further addition of 1 µmol/L SNAP or
1 µmol/L DEA/NO resulted in a significant enhancement of contractile
activation.
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Discussion |
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TopAbstractIntroductionMaterials and Methods Results Discussion References |
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We investigated the effects of organic nitrates and spontaneous NO donors on the basal level of cGMP and on the contractile function of isolated rat ventricular myocytes. It is the major finding that high concentrations of organic nitrates and low concentrations of spontaneous NO donors cause a small increase in cellular cGMP level and, concurrently, a pronounced increase in contractile response. The contractile effect of isoprenaline is enhanced under these conditions. In contrast, high concentrations of spontaneous NO donors elicit a pronounced increase in cellular cGMP content and depress the contractile response of cardiomyocytes.
In the present study, not only spontaneous NO donors like SNAP and
DEA/NO but also organic nitrates like GTN, ISMN, and PETN increased the
basal level of cGMP in rat ventricular myocytes. According
to the present knowledge, organic nitrates like GTN and ISMN are
prodrugs,2 3 a fact that has also been demonstrated
for
PETN.4 Their active principle, NO, is produced by a
yet-unknown metabolic pathway occurring in the vascular
wall.5 6 Our results indicate that this
metabolic pathway also exists in
cardiomyocytes, since basal levels of cGMP increased
significantly in the presence of organic nitrates (Figs 1 and
2).
Compared with aortic ring preparations of the same animals, however,
the activity of the organic nitrate GTN to increase cGMP content was
low in cardiomyocytes (40-fold increase over basal cGMP
[Table 1] versus a twofold increase over basal cGMP
[Fig 2] for
aortic rings versus cardiomyocytes, respectively). A
similar difference between cardiomyocytes and aortic rings
was observed in experiments using the spontaneous NO donor SNAP (Fig
1,
Table 1). In cardiomyocytes, 100 µmol/L SNAP induced an
eightfold increase in cGMP, whereas in aortic rings a 160-fold increase
was observed. Both results indicate that an increase in cGMP initiated
by NO, whether derived from organic nitrates or spontaneous NO donors,
is much more pronounced in vascular tissue. These results are
consistent with those of others obtained in mammalian
tissues.1 25 There may be several reasons for this
difference between cardiac and vascular tissue, but according to the
cytosolic production and hydrolysis of cGMP, an involvement of
sGC or of the phosphodiesterases responsible for cGMP hydrolysis seems
to be most probable.1 However, in aortic rings and in
cardiomyocytes, SNAP induced an approximately fourfold
stronger increase in basal cGMP than did GTN (Fig 2, Table
1). This
similar fourfold difference in maximal cGMP production
initiated by GTN and SNAP indicates a comparable production of
NO from organic nitrates in these tissues. Thus, treatment with organic
nitrates in vivo probably increases cGMP in cardiac muscle.
Spontaneous NO donors and organic nitrates exerted profound effects on
the contractile response of ventricular
cardiomyocytes to electrical field stimulation, as
evaluated by investigating changes in cell shortening. Focusing on
functional consequences of increased cGMP in ventricular
muscle, several previous investigations showed a depression of
contractile function in frog and mammalian cardiac
muscle.13 14 26 27 A similar
result was observed in the
present study after incubation of electrically stimulated rat
ventricular myocytes with high concentrations of SNAP and
DEA/NO (Fig 4C and 4D), which both markedly
increased cGMP levels (Fig 2). Previous studies provided
evidence that cGMP inhibits transmembrane
calcium current.10 15 28 29
In mammalian cardiac myocytes,
this effect of cGMP is mediated by the activation of cGMP-dependent
protein kinase.11 Therefore, we expected that incubation
of ventricular myocytes with KT5823, a relatively specific
inhibitor of cGMP-dependent protein kinase,30
would blunt the effect of high concentrations of NO donors. This was
indeed the case, but preincubation with KT5823 not only abolished the
inhibitory contractile effect of 100 µmol/L SNAP but also
converted this depressant action into a pronounced enhancement of
contractile response (Fig 5B). A similar result was obtained by
using
10 µmol/L of SNAP (Fig 5A). Further studies revealed that
treatment
with KT5823 unmasked a stimulating effect of NO donors on the
contractile response of electrically stimulated
cardiomyocytes, which could also be demonstrated by using a
100-fold lower concentration of SNAP and DEA/NO alone (Fig 4C
and 4D).
One might expect that in the presence of KT5823, a low concentration of
SNAP (ie, 1 µmol/L) would have a distinctly more positive contractile
effect, but the increase in contractile response was not statistically
different. Presumably, the positive effect of SNAP and DEA/NO on
cardiomyocyte contraction is also based on an action of
cGMP, because it was paralleled by low but significant
elevations of cGMP levels (Fig 2). This suggestion is supported
by the
fact that the incubation of cardiomyocytes with organic
nitrates, used in a concentration producing comparably small increases
in cGMP (Fig 2), also exerted an improvement of the contractile
response, as indicated by a significant increase in cell shortening
after 90 seconds of incubation (Fig 4A and 4B).
To gain more detailed insight into the mechanism by which cGMP may
mediate this improvement of contractile response, we measured cAMP
levels after the treatment of the cardiomyocytes with GTN
(100 µmol/L) or SNAP (1 µmol/L) compared with isoprenaline (10
nmol/L) and found a comparable increase in cAMP induced by these drugs
(Fig 3). In addition, some experiments with electrically
stimulated
cardiomyocytes shown in Fig 4 were repeated in the presence
of Rp-cAMPS, a specific inhibitor of cAMP-dependent
protein kinase.31 As shown in Fig 6A and
6B, preincubation
of the cells with Rp-cAMPS completely abolished the improvement of
contractile responses of cardiomyocytes initiated by 100
µmol/L GTN or 1 µmol/L SNAP. In addition, Rp-cAMPS also
abolished the pronounced improvement of the contractile activity
induced by 100 µmol/L SNAP in the presence of KT5823 (Fig
6C). Thus,
the positive action of GTN and SNAP on the contractile response of
cardiomyocytes most likely involves a cAMP-dependent
pathway. To further investigate an involvement of this mechanism in the
effects observed, we tested whether or not a low concentration of SNAP
(1 µmol/L) affects the activity of isoprenaline in our system. The
result of this experiment (Fig 7) supports our suggestion,
since 1
µmol/L SNAP significantly enhanced the increase in the contractile
response due to treatment with 10 nmol/L isoprenaline. The
difference between the contractile stimulatory effects of 10
nmol/L isoprenaline and 1 µmol/L SNAP was larger than expected from
the respective changes in cAMP contents (Figs 3 and
7). This could be
due, among other possibilities, to nonlinearity of the
cAMP–contractile response relation or differences in intracellular
cAMP compartmentation. It seems less likely that cGMP-dependent
inhibition blunts part of the cAMP-mediated response to 1 µmol/L
SNAP. This is because blockade of such a cGMP-dependent pathway by
KT5823 did not cause a significant change in the contractile response
to 1 µmol/L SNAP (see "Results").
Although the present study does not provide direct evidence of the
mechanisms of action by which cGMP affects cAMP levels, we suggest that
it is due to a cGMP-dependent inhibition of cAMP hydrolysis by the
cGMP-inhibited phosphodiesterase. This enzyme, which exerts a high
specificity for the hydrolysis of cAMP, is present in large amounts
in the mammalian heart,32 including
ventricular tissue of the rat.33 In previous
reports, a similar mechanism was proposed to explain the synergistic
effects of cAMP and cGMP, causing an inhibition of platelet
aggregation34 or promoting calcium currents in
ventricular myocytes.15 According to the
latter investigation, it seems likely that the suggested inhibition of
cAMP degradation by cGMP enhances a cAMP-dependent stimulation of
transmembrane calcium flux. Enhanced transmembrane calcium flux also
occurred in intact ventricular cardiomyocytes
from frogs and guinea pigs as a result of treatment with
3-morpholinosydnonimine.35 36 Our suggestion that
inhibition of cAMP hydrolysis by the cGMP-inhibited phosphodiesterase
is responsible for the positive effects of NO donors on the
contractions of ventricular cardiomyocytes is
also consistent with the reported lack of effect of
8-bromo-cGMP, even in the presence of
KT5823,10 13 37
because 8-bromo-cGMP is a 10-fold stronger stimulator of
cGMP-dependent protein kinase but a 60-fold weaker
inhibitor of cGMP-inhibited phosphodiesterase compared with
cGMP.38 A summary of the proposed mechanisms of the action
of organic nitrates and spontaneous NO donors in cardiac muscle is
given in Fig 8. This scheme focuses on cGMP-dependent
nitrate actions. Nevertheless, cGMP-independent effects of these drugs
cannot be completely excluded.
|
At present, it is difficult to consider a possible
physiological or pathological significance of the
reported biphasic activity of NO (and thus cGMP) on the contractile
response of isolated cardiomyocytes. In the present
study, a low concentration of SNAP significantly improved the activity
of isoprenaline on the contractile force of isolated rat left
ventricular papillary muscle (Table 2), indicating the
existence of the proposed mechanism in a multicellular preparation
stimulated with a physiological driving frequency.
In accordance, a direct positive effect of sodium nitroprusside on
contractile force was observed in cat atrial strips,39 and
a similar effect occurred in cat papillary muscle without
endothelium treated with sodium
nitroprusside.40 However, in isolated guinea pig heart
(not stimulated with a ß-mimetic agent), a positive effect of
sodium nitroprusside on contractile force was not
observed.41 Similarly, intracoronary sodium
nitroprusside did not increase dP/dtmax in humans at
rest.42 On the other hand, it has been demonstrated that
the inhibition of endogenous NO production achieved
by perfusion with
NG-monomethyl-L-arginine,
decreases contractility in isolated rat hearts
stimulated with 10 nmol/L isoprenaline.43 Furthermore,
inhibition of NO synthesis in dogs in vivo was reported to be
associated with a decreased contractile function44 or a
decreased cardiac output,45 and both effects could not be
attributed solely to changes in vascular resistance. The latter results
suggest that the endogenous production of NO
participates in cardiac function in vitro and in vivo, presumably by
supporting cardiac contractility. This effect might be
due to the cGMP-activated cAMP-dependent pathway reported here
(Fig 8). The presence of NO synthase in rat ventricular
myocytes and the significant role of endogenously produced
NO in their response to carbachol was recently
evaluated.46 47 In one report, calcium-dependent as
well as calcium-independent NO synthase activity has been
determined in human cardiac tissue damaged by dilated
cardiomyopathy.48 Since
calcium-independent NO synthase produces large amounts of
NO,49 it might be speculated that the resulting high
content of cGMP in cardiomyocytes causes depression of
contractile activity, as depicted in Fig 8. According to the
cited
reports, we suggest that the concentration of cGMP in cardiac muscle
may play a physiological as well as pathological
role in cardiac function. The proposed mechanism of action shown in Fig
8 also indicates that effects induced by any change of cGMP
level in
heart muscle are directly dependent on the preexisting concentration of
cGMP. In addition, we suggest that the therapeutic value of organic
nitrates may not be solely due to their vascular actions but may also
involve a direct action on heart muscle.
In summary, the results of the present study demonstrate that organic nitrates and nitrovasodilators spontaneously releasing NO evoke two different and functionally opposing effects in mammalian left ventricular cardiomyocytes, being directly dependent on their influence on cGMP synthesis. A small increase in basal cGMP level initiated by either of the drugs predominantly improves the contractile response of cardiomyocytes, increases the cAMP level, and enhances the activity of ß-agonists like isoprenaline. The latter effect was also present in rat left ventricular papillary muscle. This action is presumably mediated by the inhibition of cGMP-dependent phosphodiesterase and the activation of cAMP-dependent protein kinase. By contrast, at a high level of cGMP, which was solely achieved with high doses of spontaneous NO donors, this positive contractile effect is superimposed by a depressant action on cardiomyocyte contraction, presumably mediated by activation of cGMP-dependent protein kinase. Finally, the present study demonstrates bioactivation of organic nitrates in left ventricular cardiomyocytes, suggesting a direct effect of these drugs on heart muscle.
|
Selected Abbreviations and Acronyms |
|---|
|
|
Acknowledgments |
|---|
This study was supported by Deutsche Forschungsgemeinschaft, SFB 242 (Projekt A/11). The authors wish to thank Dr L. Keefer (National Cancer Institute, Frederick, Md) for providing DEA/NO, Dr D. Stalleicken (ISIS-Pharma, Zwickau, FRG) for providing ISMN and PETN, Dr Sandrock (Schwarz Pharma AG, Monheim, FRG) for providing GTN, and Dr D. Hafner (Institut für Pharmakologie, Heinrich-Heine Universität, Düsseldorf, FRG) for supplying computer programs used for calculation of statistics and figures.
|
Footnotes |
|---|
Previously presented in abstract form at the 67th Scientific Sessions of the American Heart Association in Dallas, Tex, November 14-17, 1994 (Circulation. 1994;90[suppl 1]:I-592).
Received May 22, 1995; accepted September 12, 1995.
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References |
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D. Sarkar, P. Vallance, and S. E. Harding Nitric oxide: not just a negative inotrope Eur J Heart Fail, October 1, 2001; 3(5): 527 - 534. [Abstract] [Full Text] [PDF]
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 M. O. Stojanovic, M. T. Ziolo, G. M. Wahler, and B. M. Wolska Anti-adrenergic effects of nitric oxide donor SIN-1 in rat cardiac myocytes Am J Physiol Cell Physiol, July 1, 2001; 281(1): C342 - C349. [Abstract] [Full Text] [PDF]
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S. M Bryant, C. E Sears, L. Rigg, D. A Terrar, and B. Casadei Nitric oxide does not modulate the hyperpolarization-activated current, If, in ventricular myocytes from spontaneously hypertensive rats Cardiovasc Res, July 1, 2001; 51(1): 51 - 58. [Abstract] [Full Text] [PDF]
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G. Vandecasteele, I. Verde, C. Rucker-Martin, P. Donzeau-Gouge, and R. Fischmeister Cyclic GMP regulation of the L-type Ca2+ channel current in human atrial myocytes J. Physiol., June 1, 2001; 533(2): 329 - 340. [Abstract] [Full Text] [PDF]
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A. Godecke, T. Heinicke, A. Kamkin, I. Kiseleva, R. H Strasser, U. K M Decking, T. Stumpe, G. Isenberg, and J. Schrader Inotropic response to {beta}-adrenergic receptor stimulation and anti-adrenergic effect of ACh in endothelial NO synthase-deficient mouse hearts J. Physiol., April 1, 2001; 532(1): 195 - 204. [Abstract] [Full Text] [PDF]
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N. Abi-Gerges, R. Fischmeister, and P.-F. Mery G protein-mediated inhibitory effect of a nitric oxide donor on the L-type Ca2+ current in rat ventricular myocytes J. Physiol., February 15, 2001; 531(1): 117 - 130. [Abstract] [Full Text] [PDF]
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D. Sarkar, P. Vallance, C. Amirmansour, and S. E. Harding Positive inotropic effects of NO donors in isolated guinea-pig and human cardiomyocytes independent of NO species and cyclic nucleotides Cardiovasc Res, December 1, 2000; 48(3): 430 - 439. [Abstract] [Full Text] [PDF]
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N. Paolocci, U. E. G. Ekelund, T. Isoda, M. Ozaki, K. Vandegaer, D. Georgakopoulos, R. W. Harrison, D. A. Kass, and J. M. Hare cGMP-independent inotropic effects of nitric oxide and peroxynitrite donors: potential role for nitrosylation Am J Physiol Heart Circ Physiol, October 1, 2000; 279(4): H1982 - H1988. [Abstract] [Full Text] [PDF]
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G. Heusch, H. Post, M. C. Michel, M. Kelm, and R. Schulz Endogenous Nitric Oxide and Myocardial Adaptation to Ischemia Circ. Res., July 21, 2000; 87(2): 146 - 152. [Abstract] [Full Text] [PDF]
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L. Sandirasegarane, R. Charles, N. Bourbon, and M. Kester NO regulates PDGF-induced activation of PKB but not ERK in A7r5 cells: implications for vascular growth arrest Am J Physiol Cell Physiol, July 1, 2000; 279(1): C225 - C235. [Abstract] [Full Text] [PDF]
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G. Muller-Strahl, K. Kottenberg, H.-G. Zimmer, E. Noack, and G. Kojda Inhibition of nitric oxide synthase augments the positive inotropic effect of nitric oxide donors in the rat heart J. Physiol., January 15, 2000; 522(2): 311 - 320. [Abstract] [Full Text] [PDF]
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J. G. Lainchbury, J. C. Burnett Jr., D. Meyer, and M. M. Redfield Effects of natriuretic peptides on load and myocardial function in normal and heart failure dogs Am J Physiol Heart Circ Physiol, January 1, 2000; 278(1): H33 - H40. [Abstract] [Full Text] [PDF]
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 Y. Tanoue, S. Morita, Y. Ochiai, N. Haraguchi, R. Tominaga, Y. Kawachi, and H. Yasui NITROGLYCERIN AS A NITRIC OXIDE DONOR ACCELERATES LIPID PEROXIDATION BUT PRESERVES VENTRICULAR FUNCTION IN A CANINE MODEL OF ORTHOTOPIC HEART TRANSPLANTATION J. Thorac. Cardiovasc. Surg., September 1, 1999; 118(3): 547 - 556. [Abstract] [Full Text] [PDF]
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W. J Paulus and A. M Shah NO and cardiac diastolic function Cardiovasc Res, August 15, 1999; 43(3): 595 - 606. [Full Text] [PDF]
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J.-L. Balligand Regulation of cardiac {beta}-adrenergic response by nitric oxide Cardiovasc Res, August 15, 1999; 43(3): 607 - 620. [Full Text] [PDF]
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J.-M. Chesnais, R. Fischmeister, and P.-F. Mery Positive and negative inotropic effects of NO donors in atrial and ventricular fibres of the frog heart J. Physiol., July 15, 1999; 518(2): 449 - 461. [Abstract] [Full Text] [PDF]
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M. G. Vila-Petroff, A. Younes, J. Egan, E. G. Lakatta, and S. J. Sollott Activation of Distinct cAMP-Dependent and cGMP-Dependent Pathways by Nitric Oxide in Cardiac Myocytes Circ. Res., May 14, 1999; 84(9): 1020 - 1031. [Abstract] [Full Text] [PDF]
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G. J Crystal and X. Zhou Nitric oxide does not modulate the increases in blood flow, O2 consumption, or contractility during CaCl2 administration in canine hearts Cardiovasc Res, April 1, 1999; 42(1): 232 - 239. [Abstract] [Full Text] [PDF]
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G. Kojda and K. Kottenberg Regulation of basal myocardial function by NO Cardiovasc Res, March 1, 1999; 41(3): 514 - 523. [Full Text] [PDF]
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N. Suto, A. Mikuniya, T. Okubo, H. Hanada, N. Shinozaki, and K. Okumura Nitric oxide modulates cardiac contractility and oxygen consumption without changing contractile efficiency Am J Physiol Heart Circ Physiol, July 1, 1998; 275(1): H41 - H49. [Abstract] [Full Text] [PDF]
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D. L. Brutsaert, P. Fransen, L. J. Andries, G. W. De Keulenaer, and S. U. Sys Cardiac endothelium and myocardial function Cardiovasc Res, May 1, 1998; 38(2): 281 - 290. [Abstract] [Full Text] [PDF]
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P. A De Mulder, S. G De Hert, R. J Van Kerckhoven, H. F Adriaensen, and T. C Gillebert Sodium nitroprusside enhances in vivo left ventricular function in {beta}-adrenergically stimulated rabbit hearts Cardiovasc Res, April 1, 1998; 38(1): 133 - 139. [Abstract] [Full Text] [PDF]
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 G. Kojda, M. Patzner, A. Hacker, and E. Noack Nitric Oxide Inhibits Vascular Bioactivation of Glyceryl Trinitrate: A Novel Mechanism to Explain Preferential Venodilation of Organic Nitrates Mol. Pharmacol., March 1, 1998; 53(3): 547 - 554. [Abstract] [Full Text]
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R. E Klabunde, J. Tse, and H. R Weiss Guanylyl cyclase inhibition reduces contractility and decreases cGMP and cAMP in isolated rat hearts Cardiovasc Res, March 1, 1998; 37(3): 676 - 683. [Abstract] [Full Text] [PDF]
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P. C. Kouretas, A. K. Myers, Y. D. Kim, P. A. Cahill, J. L. Myers, Y.-N. Wang, J. V. Sitzmann, R. B. Wallace, and R. L. Hannan Heparin And Nonanticoagulant Heparin Preserve Regional Myocardial Contractility After Ischemia-Reperfusion Injury: Role Of Nitric Oxide J. Thorac. Cardiovasc. Surg., February 1, 1998; 115(2): 440 - 449. [Abstract] [Full Text] [PDF]
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J. M. Hare, M. M. Givertz, M. A. Creager, and W. S. Colucci Increased Sensitivity to Nitric Oxide Synthase Inhibition in Patients With Heart Failure : Potentiation of ß-Adrenergic Inotropic Responsiveness Circulation, January 20, 1998; 97(2): 161 - 166. [Abstract] [Full Text] [PDF]
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H. Hu Nipavan Chiamvimonvat Toshio Yamagishi Eduardo Direct Inhibition of Expressed Cardiac L-Type Ca2+ Channels by S-Nitrosothiol Nitric Oxide Donors Circ. Res., November 19, 1997; 81(5): 742 - 752. [Abstract] [Full Text]
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W. J. Paulus, S. Kastner, P. Pujadas, A. M. Shah, H. Drexler, and M. Vanderheyden Left Ventricular Contractile Effects of Inducible Nitric Oxide Synthase in the Human Allograft Circulation, November 18, 1997; 96(10): 3436 - 3442. [Abstract] [Full Text]
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B. Preckel, G. Kojda, W. Schlack, D. Ebel, K. Kottenberg, E. Noack, V. Thamer, E. Noack, and V. Thämer Inotropic Effects of Glyceryl Trinitrate and Spontaneous NO Donors in the Dog Heart Circulation, October 21, 1997; 96(8): 2675 - 2682. [Abstract] [Full Text]
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 J.-L. Balligand and P. J. Cannon Nitric Oxide Synthases and Cardiac Muscle : Autocrine and Paracrine Influences Arterioscler. Thromb. Vasc. Biol., October 1, 1997; 17(10): 1846 - 1858. [Abstract] [Full Text]
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M. Straznicka, G. Gong, J. Tse, P. M. Scholz, and H. R. Weiss cGMP level that reduces cardiac myocyte O2 consumption is altered in renal hypertension Am J Physiol Heart Circ Physiol, October 1, 1997; 273(4): H1949 - H1955. [Abstract] [Full Text] [PDF]
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A. J. Sherman, C. A. Davis III, F. J. Klocke, K. R. Harris, G. Srinivasan, A. S. Yaacoub, D. A. Quinn, K. A. Ahlin, and J. J. Jang Blockade of Nitric Oxide Synthesis Reduces Myocardial Oxygen Consumption In Vivo Circulation, March 4, 1997; 95(5): 1328 - 1334. [Abstract] [Full Text]
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J. Bartunek, A. M. Shah, M. Vanderheyden, and W. J. Paulus Dobutamine Enhances Cardiodepressant Effects of Receptor-Mediated Coronary Endothelial Stimulation Circulation, January 7, 1997; 95(1): 90 - 96. [Abstract] [Full Text]
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R. A. Kelly, J.-L. Balligand, and T. W. Smith Nitric Oxide and Cardiac Function Circ. Res., September 1, 1996; 79(3): 363 - 380. [Full Text]
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N. Abi-Gerges, G. Szabo, A. S. Otero, R. Fischmeister, and P.-F. Mery NO donors potentiate the {beta}-adrenergic stimulation of ICa,L and the muscarinic activation of IK in rat cardiac myocytes J. Physiol., February 22, 2002; (2002) 200101292. [Abstract] [PDF]
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U. Laber, T. Kober, V. Schmitz, A. Schrammel, W. Meyer, B. Mayer, M. Weber, and G. Kojda Effect of Hypercholesterolemia on Expression and Function of Vascular Soluble Guanylyl Cyclase Circulation, February 19, 2002; 105(7): 855 - 860. [Abstract] [Full Text] [PDF]
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F. Brunner, P. Andrew, G. Wolkart, R. Zechner, and B. Mayer Myocardial Contractile Function and Heart Rate in Mice With Myocyte-Specific Overexpression of Endothelial Nitric Oxide Synthase Circulation, December 18, 2001; 104(25): 3097 - 3102. [Abstract] [Full Text] [PDF]
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