© 1999 American Heart Association, Inc.
Original Contributions |
Activation of Distinct cAMP-Dependent and cGMP-Dependent Pathways by Nitric Oxide in Cardiac Myocytes
From the Laboratory of Cardiovascular Science (M.G.V.-P., A.Y., E.G.L., S.J.S.) and Laboratory of Clinical Investigation (J.E.), Intramural Research Program, National Institute on Aging, Gerontology Research Center, Baltimore, Md.
Correspondence to Steven J. Sollott, MD, Laboratory of Cardiovascular Science, Gerontology Research Center, Box 13, Intramural Research Program, National Institute on Aging, 5600 Nathan Shock Dr, Baltimore, MD 21224-6825. E-mail sollotts{at}grc.nia.nih.gov
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Abstract—Nitric oxide (NO) donors were recently shown to produce biphasic contractile effects in cardiac tissue, with augmentation at low NO levels and depression at high NO levels. We examined the subcellular mechanisms involved in the opposing effects of NO on cardiac contraction and investigated whether NO modulates contraction exclusively via guanylyl cyclase (GC) activation or whether some contribution occurs via cGMP/PKG-independent mechanisms, in indo 1–loaded adult cardiac myocytes. Whereas a high concentration of the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 100 µmol/L) significantly attenuated contraction amplitude by 24.4±4.5% (without changing the Ca2+ transient or total cAMP), a low concentration of SNAP (1 µmol/L) significantly increased contraction amplitude (38±10%), Ca2+ transient (26±10%), and cAMP levels (from 6.2 to 8.5 pmol/mg of protein). The negative contractile response of 100 µmol/L SNAP was completely abolished in the presence of the specific blocker of PKG KT 5823 (1 µmol/L); the positive contractile response of 1 µmol/L SNAP persisted, despite the presence of the selective inhibitor of GC 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10 µmol/L) alone, but was completely abolished in the presence of ODQ plus the specific inhibitory cAMP analog Rp-8-CPT-cAMPS (100 µmol/L), as well as by the NO scavenger oxyhemoglobin. Parallel experiments in cell suspensions showed significant increases in adenylyl cyclase (AC) activity at low concentrations (0.1 to 1 µmol/L) of SNAP (AC, 18% to 20% above basal activity). We conclude that NO can regulate both AC and GC in cardiac myocytes. High levels of NO induce large increases in cGMP and a negative inotropic effect mediated by a PKG-dependent reduction in myofilament responsiveness to Ca2+. Low levels of NO increase cAMP, at least in part, by a novel cGMP-independent activation of AC and induce a positive contractile response.
Key Words: nitric oxide signaling • cGMP • cAMP • contractility • cardiac myocyte
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Introduction |
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Nitric oxide (NO) has been implicated as a mediator of many cellular processes, including endothelium-dependent relaxation of blood vessels, chemical communication between peripheral nerves and smooth muscle, inhibition of platelet aggregation, immune responses, and neurotransmission.1 These effects of NO have been ascribed to the activation of soluble guanylyl cyclase (GC), leading to the production of cGMP and activation of cGMP-dependent protein kinase (PKG).1 2 Furthermore, NO and NO donors have been shown to elicit a wide range of effects on myocardial contractility,3 4 5 6 7 8 9 but the specific nature of the subcellular mechanisms underlying the diverse effects in heart is largely unknown.
Although the coronary endothelium is responsible for the bulk of the endogenous, physiological production of NO in the heart,10 11 NO can also be produced within the cardiac myocytes themselves (ie, autocrine production) by the constitutive NO synthase NOS-3.12 13 Dynamic regulation of NO production in the heart apparently yields beat-to-beat oscillations in response to changes in coronary flow and myocardial loading and achieves micromolar levels in close vicinity to the cardiac myocyte.11 Until recently, it was generally accepted that NO induced negative inotropic effects in cardiac preparations, mediated principally through cGMP-related mechanisms, specifically via the reduction of Ca2+ influx through L-type Ca2+ channels, either through activation of cGMP-dependent phosphodiesterase14 15 16 or PKG and/or phosphatases.17 18 19 20 cGMP has also been shown to decrease relative myofilament response to Ca2+ and therefore enhances myocardial relaxation and reduces diastolic tone.21 However, the hypothesis that cGMP-mediated processes are the only mechanisms responsible for the NO-mediated contractile effects has subsequently been challenged. Several studies have demonstrated a dissociation between cGMP concentrations and contractile state, because low concentrations of acetylcholine induced a negative inotropic effect even in the absence of any change in cGMP concentration.22 23 24 25 Recent reports show that under certain conditions, NO donors are able to enhance myocardial contractility,8 9 and that the basal intracardiac production and release of NO significantly augments the Frank-Starling response in the isolated heart.26 It has been demonstrated that the augmentation of contractility by exogenous NO could be the result of an elevation of the intracellular levels of cAMP due to the cGMP-dependent inhibition of cAMP hydrolysis by cGMP-inhibited phosphodiesterase (cGI-PDE or PDE type III).9 These observations, however, do not rule out the possibility of cAMP being increased by a direct (ie, GC/cGMP independent) modulation of the adenylyl cyclase (AC)/cAMP/cAMP-dependent protein kinase (PKA) pathway by NO.
Most reports on NO-mediated regulation of cardiac excitation and contraction have focused on the cGMP-dependent actions, whereas GC/cGMP-independent mechanisms have been largely unexplored. Relatively recently, a few publications have addressed "unconventional" pathways for NO signaling in the heart whereby NO may directly modulate protein function in a GC/cGMP-independent fashion, eg, via trans-nitrosylation of critical or regulatory thiols.27 28 29 Although NO certainly modulates cardiac contraction in part via cGMP/PKG-related mechanisms, considering the multiplicity of potential NO targets in the heart and the multifaceted effects of NO and NO-related compounds on cardiac contraction, it seems unlikely that NO exerts all of its physiologically relevant effects exclusively through activation of GC and PKG activation.
To examine whether NO potentially modulates the cAMP/PKA signaling pathway via GC-independent mechanisms, we studied the effects of the NO donor S-nitroso-N-acetylpenicillamine (SNAP), in the presence and absence of specific inhibitors of GC, PKG, and PKA, on contraction and the Ca2+ transient (Cai) in isolated rat cardiac myocytes, in parallel with measurements of intracellular cGMP, cAMP, and AC activity. Parallel functional experiments were performed with 3-[2-hydroxy-1-(1-methylethyl)-2-nitrosohydrazino]-1-propanamine (NOC5), an NO amine complex that serves as an NO donor chemically distinct from SNAP, and with NO scavengers, to establish that the biological effects were the results of the action of NO. The results from the present study suggest that NO is capable of evoking either positive or negative inotropic responses in cardiac myocytes depending on the concentration of exposure. Whereas the decrease in contractile response seen principally at higher levels of NO can be attributed mainly to a cGMP-dependent reduction in myofilament responsiveness to Ca2+, the enhanced contractile response at lower NO levels is due to increased intracellular cAMP levels that are mediated, at least in part, through a novel NO-dependent, GC/cGMP-independent activation of AC. Preliminary findings of the present study have been previously published in abstract form.30 31
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Materials and Methods |
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Cardiac Myocyte Isolation
Single cardiac myocytes were isolated via a previously described technique with minor modifications.32 Briefly, 2- to 4-month-old adult Sprague-Dawley rats (Charles River) were anesthetized with intraperitoneal sodium pentobarbital, and hearts were rapidly excised and perfused retrogradely with 25 mL nominally Ca2+-free bicarbonate buffer of the following composition (in mmol/L): NaCl 116.4, KCl 5.4, MgSO4 1.2, NaH2PO4 1.2, glucose 5.6, and NaHCO3 26.2, kept at 36±1°C and continuously gassed with 95% O2 and 5% CO2 to keep pH at 7.4. Perfusion was continued with a similar bicarbonate buffer containing 50 µmol/L Ca2+, 0.1% collagenase type B, 0.04 mg/mL protease XVI, and 0.1% BSA type V for
20 minutes. The left
ventricle was then mechanically dissociated, and myocytes were
resuspended in a series of HEPES buffers with gradually increasing
Ca2+ concentration. Cells were finally suspended
in HEPES buffer containing (in mmol/L) NaCl 137, KCl 4.9,
MgSO4 1.2,
NaH2PO4 1.2, glucose 15,
HEPES 20, and CaCl2 1.0, (pH 7.4) and stored at
room temperature until use. Noncardiac myocytes comprised <<1% of
cells prepared in this manner. Cardiac myocyte viability was typically
70% to 80%. All experiments were replicated at least 3 times (or as
specifically stated) and represent cells from at least 2 (or
more) different myocyte preparations. Experiments were performed under
a protocol approved by the National Institute on Aging Animal Care
Committee.
Simultaneous Measurements of Contraction and Indo
1 Fluorescence
Changes in the Cai, assessed by indo 1
fluorescence, and cell length were measured in isolated
myocytes as previously described.33 In brief, for
measurements of [Ca2+]i,
myocytes were loaded with the membrane-permeable acetoxymethyl ester of
indo 1 (indo 1-AM), using a 10-minute exposure to a 25 µmol/L
solution in HEPES buffer at room temperature. After loading, cells were
transferred to a Lucite chamber with a glass coverslip on the stage of
an inverted microscope and were continuously superfused with HEPES
buffer. Myocytes were chosen for study according to previously
established criteria,32 ie, a rod-shaped appearance with
clear striations and no membrane blebs, a negative staircase of twitch
performance on stimulation from rest, and the absence of
spontaneous contractions. Cardiac myocyte contraction was produced via
electrical field stimulation from 2 platinum electrodes (2- to 4-ms
duration at 0.5 Hz) connected to a stimulator (model SD9, Grass
Instrument Co).
Myocyte length at rest and during contraction was monitored from a red light (650 to 750 nm) bright field image of the cell projected onto a photodiode array (Reticon) with a 5-ms temporal resolution. For simultaneous measurement of the Cai in these cells, indo 1 fluorescence was excited by epi-illumination with 10-µs flashes of 350±5 nm of light focused onto the myocyte by a 100x/1.3NA Nikon UV fluor glycerine immersion objective. Epifluorescence collected every 5 ms was split into 391- to 434-nm (410 channel) and 457- to 507-nm (490 channel) wavelength bands by bandpass interference filters. The ratio of the indo 1 emission at the 2 wavelengths was calculated using a pair of fast integrator sample-and-hold circuits under the control of a specially modified PC, and it was taken as an index of the Cai. When isolated cardiac myocytes are loaded with indo 1-AM, there is a variable compartmentalization of the indicator into the mitochondria33 that prevents the use of a standard calibration curve. Thus, the present results in indo 1-AM–loaded myocytes will be expressed as fluorescence ratio rather than as absolute [Ca2+]i values. Both loading of the Ca2+ probe into the myocytes and the experiments were performed at 25°C to minimize the loss of the fluorescent Ca2+ indicator from the cell.33 Additionally, some experiments were implemented with cells that had not been loaded with indo 1, and only cell length was measured.
Assessment of Myofilament Response to Ca2+
Changes in myofilament responsiveness to
Ca2+ were assessed using the steady-state
relation between cell length and
[Ca2+]i in intact single
cardiac myocytes tetanized by high-frequency (10 Hz) stimulation after
exposure to thapsigargin (0.2 µmol/L for 15 minutes), as
described previously.34 Thapsigargin disables the
sarcoplasmic reticulum35 and thus enables tetanization of
otherwise intact myocytes. High-frequency electrical stimulation after
thapsigargin treatment results in the effective summation of the
repetitive transmembrane flux of Ca2+ current
(because of the absence of sarcoplasmic reticulum
Ca2+ sequestration and periodic release) to
achieve a steady-state level of myoplasmic Ca2+
substantially elevated above resting levels, at the point that
Ca2+ influx is balanced by the rate of
Ca2+ extrusion (via Na/Ca exchange). With this
approach, the Cai can be reversibly clamped near
peak systolic levels during the tetanic contracture for 10 to
60 seconds (or longer) and then rapidly returned to resting levels on
cessation of electrical stimulation via normal Na/Ca exchange
mechanisms. The steady-state levels of Ca2+
achieved during tetanization, moreover, can be systematically regulated
by adjustments in the concentration of bathing
Ca2+ and can be matched between protocols to
effectively gauge changes in myofilament Ca2+
sensitivity. Thus, changes in the degree of cell shortening between
tetani clamped at the same Cai level can be
attributed to changes in the relative myofilament responsiveness to
Ca2+.
Determination of cGMP and cAMP
Suspensions of freshly isolated adult rat cardiac myocytes were
pretreated for 30 minutes at 23°C with either control or
1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) 10 µmol/L
containing buffers and subsequently challenged with the indicated
concentration of SNAP for the next 20 minutes. The cells were then
lysed with 1 mL ice-cold 0.6 mmol/L perchloric acid. The cell
lysates (950 µL) were transferred to microcentrifuge tubes,
and the pH was adjusted to 7.0 using
K2CO3. After
centrifugation for 5 minutes at 8000g, the
supernatant was vacuum-dried and then recovered in 200 µL Tris/EDTA
buffer. After addition of 0.15 mmol/L
Na2CO3 (20 µL) and
0.15 mmol/L ZnSO4 (20 µL) and incubation
for 15 minutes on ice, the salt precipitate was removed by
centrifugation for 5 minutes at 1200g, and
50 µL of supernatant was assayed using either a cAMP
[3H] or cGMP [3H] assay
kit (Amersham). Cellular protein was measured using the Bradford method
(Bio-Rad) with bovine 
-globulin as standard.
Determination of AC Activity
Preparation of Purified Cardiac Sarcolemmal Membranes
After mincing and washing with saline EGTA (154 mmol/L
NaCl, 0.1 mmol/L EGTA), freshly excised cardiac
ventricular tissue was suspended into 5 volumes of
homogenizing buffer (HB) (in mmol/L: HEPES 10,
MgCl2 2, EGTA 0.1, and dithiothreitol 5; prepared
the day of use). The suspension was homogenized 4 times for
10 seconds at 22 000 rpm with a polytron. Five more volumes of HB were
added to the homogenate, and after
centrifugation at 12 000g for 15 minutes at
4°C, the supernatants were poured into polypropylene
ultracentrifuge tubes and centrifuged at
50 000g for 30 minutes at 4°C in an
ultracentrifuge. The resulting pellets were then resuspended in
a motorized Potter-Elvehjem glass/Teflon homogenizer
with 0.75 HB per gram of original sample. Membrane protein was assayed
by the BCA protein assay method (Pierce), and the
homogenate was stored at -80°C until use.
AC Enzyme Activity
Two micrograms of membrane protein was added into a final volume
of 200 µL containing (in mmol/L) HEPES 25,
MgCl2 0.4, GTP 5, dithiothreitol 0.15, NaCl 50,
creatine phosphate 2.5, creatine phosphokinase 5 U,
3-isobutyl-1-methylxanthine (IBMX) 10, and alamethicin (1 µg/µg of
membrane protein). The tubes were transferred to a 30°C water bath,
and the reaction with a given dose of SNAP was started by adding 5
mmol/L ATP. After 15 minutes, the reaction was stopped by addition of
1.8 mL of 80°C to 90°C H2O. Subsequently,
cAMP was determined using the scintillation proximity assay according
to the Amersham protocol furnished with the RPA 538 kit (Amersham).
Determination of Free NO Concentration Produced by NO Donors SNAP
and NOC5
Free NO concentration was determined in the
physiological buffer solutions containing either
SNAP or NOC5 at the concentrations used in these experiments (90
minutes after preparation, 25°C) using a Sievers model 280 nitric
oxide analyzer following the method recommended by the
manufacturer (NO concentrations were also measured in the bathing
solutions of some of the cardiac myocyte contraction experiments to
ensure consistency of NO application). Calibration of the
NO analyzer was obtained using reagent-grade
NaNO3 prepared in nitrate-free, deionized water
(standard solutions containing 10 nmol/L, 50 nmol/L, and 100 nmol/L and
1 µmol/L, 5 µmol/L, 10 µmol/L, and 100
µmol/L nitrate were used for calibration), in a nitrate-reducing
reaction system of freshly prepared VCl3/HCl at
90°C as prescribed by Sievers, which results in the quantitative
conversion of nitrate (and nitrite) to NO. The resulting NO was
measured by the detection of chemiluminescence from its reaction with
ozone. Within-run and between-run reproducibility was <±5%.
Materials
Collagenase type B was purchased from
Boehringer Mannheim Corp; isoproterenol from Winthrop
Pharmaceuticals; protease XVI, N-acetylpenicillamine (NAP),
and IBMX from Sigma Chemical Co; indo 1-AM from Molecular Probes Inc;
BSA type V, bovine hemoglobin, SNAP, and NOC5 from Calbiochem Corp;
Rp-8-CPT-cAMPS from Biolog; and ODQ from Biomol Research Laboratories
Inc. All other chemicals were of the purest reagent grade
available.
Statistics
All data are presented as mean±SEM. Comparisons within
groups were made by paired and unpaired Student t test, and
values of P<0.05 were taken to indicate statistical
significance.
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Reversible Effects of SNAP on Cardiac Myocyte Contraction
We assessed the effect of different concentrations of SNAP on contraction in electrically stimulated cardiac myocytes. Figure 1A
shows representative
examples of the opposing effects of high (100 µmol/L) and low
(1 µmol/L) concentrations of SNAP on the unloaded contraction
from 2 separate cardiac myocytes. At the high concentration (top
tracing), SNAP induced a slowly evolving negative inotropic effect,
which was maximal 15 to 20 minutes after administration of the drug,
and this effect was completely reversible on washout. In contrast, low
concentrations of SNAP induced a pronounced positive inotropic effect,
which was also entirely reversible on washout. Figure 1B shows
the average data from these experiments, indicating the time course of
the opposing contractile responses induced by high and low
concentrations of SNAP. After 20 minutes of SNAP exposure, twitch
amplitude was reduced by 24±5% (n=5) at the high concentration but
was increased by 38±10% (n=5) at the low concentration. Thus, the
dynamic range of contractile regulation induced by the different
concentrations of NO used in these experiments is about two thirds of
the basal contraction amplitude.
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As control experiments, cells were exposed to the byproduct of SNAP
after release of NO, NAP, at the same concentrations as used in the
SNAP experiments. Figure 1C
shows that neither NAP at 100
µmol/L nor NAP at 1 µmol/L had any effect on myocyte
contraction, indicating that the various contractile effects seen with
SNAP do not result from the thiol byproduct after the
production of NO. Figure 1D demonstrates that the
positive contractile effect seen in these experiments was indeed the
result of NO, per se, insofar as that seen both with SNAP (1
µmol/L) and an unrelated NO donor, NOC5 (1 µmol/L), was
completely abolished by coincubation with the NO scavengers,
oxyhemoglobin (10 µmol/L), and carboxy-PTIO (0.1 mmol/L;
data not shown). The negative contractile effects seen at higher
concentrations of both SNAP (100 µmol/L) and NOC5 (20
µmol/L) are similarly abolished by both oxyhemoglobin and
carboxy-PTIO (data not shown). Similar results were obtained in at
least triplicate observations in different cells under each of the
conditions described above.
Effect of SNAP on [Ca2+]i, cGMP, and
cAMP
Using indo 1–loaded cardiac myocytes, we investigated the effect
of different concentrations of SNAP on contraction and
Cai measured simultaneously. A
representative example of the negative contractile
effect of a high concentration of SNAP (100 µmol/L) and the
associated Cai is depicted in Figure 2A. The progressive reduction in the
twitch amplitude seen here was not associated with a decrease of the
Cai, suggesting that SNAP (100 µmol/L)
reduced the relative myofilament responsiveness to
Ca2+. Also shown in the figure are the results
from parallel experiments comparing intracellular levels of cGMP and
cAMP in the presence and absence of SNAP. SNAP (100 µmol/L)
induced a large increase in cGMP (31±6% versus basal,
P<0.05) but had no significant effect on cAMP levels. In
contrast, a low concentration of SNAP (1 µmol/L) elicited a
positive contractile response that was associated with a significant
increase in the Cai amplitude (26±10%, n=5
cells, P<0.05; Figure 2B). This low concentration of
SNAP induced only a modest increase in cGMP (12±3% versus basal,
P<0.05) but produced a large increment in cAMP levels
(35±9% versus basal, P<0.05). The average effects of high
and low concentrations of SNAP on contraction and
Cai amplitude and kinetics are provided in the
Table.
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Effect of SNAP on Myofilament Responsiveness to
Ca2+
The results shown in Figure 2 suggest that high
concentrations of SNAP are associated with a diminished sensitivity of
the myofilaments to Ca2+. The effects of low
concentrations of SNAP on myofilament sensitivity to
Ca2+ are, however, more difficult to interpret,
because both the amplitude of the Cai and the
contraction change during the experimental protocol. To further
characterize the effects of SNAP on myofilament responsiveness to
Ca2+, steady-state tetanic contractures (which
enable the Cai to be reversibly maintained at
reproducible levels near that of peak systole for periods of 10 to 60
seconds) were performed in the presence and absence of SNAP. Figure 3A shows a representative
example of the effect of SNAP (100 µmol/L) on the steady-state
myofilament response to Ca2+. Despite the
achievement of similar peak
[Ca2+]i levels, in the
presence of high concentrations of SNAP, steady-state cell contraction
amplitude during the tetanus was significantly decreased, confirming
that the phenomenon seen in Figure 2A was the result of a
reduction in myofilament response to Ca2+. In
contrast, perfusion with a low concentration of SNAP (1 µmol/L)
did not significantly affect either peak steady-state
Ca2+ or shortening during the tetanus (Figure 3B), thus indicating that low concentrations of SNAP do not
exert an appreciable effect on myofilament responsiveness to
Ca2+. Similar results were obtained in at least
triplicate observations in different cells at each concentration of
SNAP.
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To assess whether the increase in cGMP induced by a high concentration
of SNAP (ie, Figure 2A, right panel) activates PKG and
is, in turn, responsible for the observed reduction in myofilament
responsiveness to Ca2+, steady-state tetanic
contractures were also performed in cardiac myocytes pretreated with
the specific blocker of PKG KT 5823 1 µmol/L. Figure 4 shows that inhibiting PKG activation
with KT 5823 completely abolished the reduction in steady-state cell
shortening during the tetany in the presence of high concentrations of
SNAP (100 µmol/L). This protocol with KT 5823 has been shown to
completely inhibit the negative contractile effect (of comparable
magnitude to that seen here with SNAP 100 µmol/L) induced by
8-bromo-cGMP (50 µmol/L) in cardiac myocytes.21
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Effect of SNAP in the Presence of Inhibitors of
GC
To investigate whether cGMP-independent mechanisms participate in
the positive inotropic effect elicited by a low concentration of SNAP,
experiments were performed in the presence of the selective
inhibitor of NO-sensitive GC36 ODQ (10
µmol/L). Figure 5A shows the effect of
SNAP (1 µmol/L) on the contraction in a
representative cardiac myocyte pretreated and
continuously perfused with ODQ. In spite of the presence of ODQ, the
positive inotropic response to SNAP (1 µmol/L) persisted
(32±7% increase in twitch amplitude) together with an increased
Cai (20±6%) (n=6 cells). Perfusion with ODQ
(10 µmol/L) alone did not significantly affect the baseline
contraction or the Cai. Figure 5C shows a
representative example of the lack of effect of SNAP
(1 µmol/L) in the presence of ODQ (10 µmol/L) on the
steady-state myofilament response to Ca2+
(similar to the result in Figure 3B), which indicates that the
positive contractile effect seen in Figure 5A is purely the
result of the increase in magnitude of the Cai
(rather than a change in myofilament Ca2+
sensitivity).
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The ability of ODQ to effectively inhibit cGMP production or to
affect cAMP levels in response to SNAP was assessed in a parallel group
of experiments in which cGMP and cAMP levels were measured in intact
cardiac myocytes in the presence and absence of ODQ (10 µmol/L).
That treatment with ODQ (10 µmol/L) was fully effective to block
GC in these cardiac myocyte experiments is demonstrated by the fact it
completely abolished the cGMP increase seen with 100 µmol/L
SNAP: specifically, SNAP (100 µmol/L) alone increased basal cGMP
(4.2±0.2 pmol/mg of protein) by 131±6%, whereas cGMP remained
98±4% of basal in the presence of SNAP (100 µmol/L)+ODQ
(P<0.05). Figure 5B shows that ODQ completely
abolished the increase in cGMP induced by SNAP 1 µmol/L, as
expected, but did not affect the increased cAMP levels. Similar effects
of ODQ on cGMP and cAMP levels were observed when higher concentrations
of SNAP were used (data not shown). Moreover, preincubation of cardiac
myocytes with ODQ was capable of transforming an otherwise typical
negative inotropic response of a high dose of SNAP (10 µmol/L)
to a sustained positive inotropic response (Figure 6). These results indicate that low
concentrations of NO are able to increase the Cai
and contraction in intact cardiac myocytes in a cGMP-independent
fashion. Although previous reports have suggested that the positive
contractile response induced by NO donors could be the result of an
elevation of the intracellular levels of cAMP (and PKA activation) due
to cGMP-dependent inhibition of PDE type III (cGMP-inhibited
phosphodiesterase or cGI-PDE),8 9 our results
indicate the presence of an important, additional pathway whereby NO
donors may increase cAMP and exert a substantial positive inotropic
action, ie, through a GC/cGMP-independent mechanism. Indeed, Figure 7 shows that even in the presence of IBMX
(0.1 mmol/L), resulting in a background of tonically inhibited
PDEs (including PDE type III), SNAP (1 µmol/L) can still exert a
positive inotropic effect through an increase of the
Cai, supporting the concept that the NO-mediated
positive inotropy seen in these experiments can occur, at least in
part, via PDE-independent mechanisms (representative of
4 cells). Because SNAP was able to elevate basal cAMP even when GC was
completely inhibited by ODQ (10 µmol/L) in these cardiac
myocytes, we hypothesized that the increase in cAMP and the positive
inotropic effect observed with low concentrations of SNAP in the
presence of ODQ could be attributed to a cGMP-independent activation of
the AC/cAMP/PKA pathway by NO.
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Effect of SNAP on AC Activity
Figure 8 illustrates the
dose-response curve for the effect of SNAP on AC activity measured on
cardiac sarcolemmal membranes. Basal AC activity was 92±6 pmol/mg of
protein per minute. Low concentrations of SNAP (0.1 and 1
µmol/L, which produced 150 nmol/L and 500 nmol/L NO, respectively)
showed significant increases of AC activity (19±6% and 16±4% above
basal activity, respectively; P<0.05). At higher SNAP
concentrations, AC activity was unchanged (at 10 µmol/L SNAP,
which produced 1.4 µmol/L NO) or reduced (by 14±6% at 100
µmol/L SNAP, which produced 14.7 µmol/L NO). The increases of
AC activity at low concentrations of SNAP (0.1 and 1 µmol/L)
were 34% to 40% of the maximal Mn2+-stimulated
AC activity.
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Effect of SNAP in the Presence of Inhibitors of GC
and PKA
To confirm that the persistent positive contractile response
induced by low concentrations of SNAP in the absence of GC activation
was mediated through cAMP-dependent PKA activation, experiments were
performed using the cAMP analog Rp-8-CPT-cAMPS previously shown to
effectively and specifically inhibit PKA-dependent
processes.37 All cells were preincubated with
Rp-8-CPT-cAMPS 100 µmol/L (at 37°C) for at least 1 hour before
the experiment. In the continuous presence of the
inhibitory cAMP analog Rp-8-CPT-cAMPS (100 µmol/L)
and ODQ (10 µmol/L), low concentrations of SNAP were unable to
elicit the positive inotropic response previously observed with SNAP
alone. Figures 9A and 9B show
representative examples of the failure of SNAP (1
µmol/L) to increase the contraction (or the associated
Cai, as in Figure 9A) of intact rat
cardiac myocytes continuously perfused with Rp-8-CPT-cAMPS with and
without ODQ, respectively (similar results were observed in 4 other
preparations). Similarly, NOC5 (1 µmol/L) also fails to elicit
an increase in contraction in the presence of Rp-8-CPT-cAMPS (±ODQ,
Figure 9C and 9D, respectively). These results demonstrate that
the positive inotropic effect of low concentrations of NO is, at least
in part, independent of cGMP and is mediated by increased cAMP and PKA
activation. In these same cells, as well as in parallel experiments,
the ability of Rp-8-CPT-cAMPS to successfully inhibit PKA activation
was routinely confirmed by its efficacy to inhibit the positive
inotropic effect induced by isoproterenol. The mean increase in
contraction amplitude induced by 1 nmol/L isoproterenol was 171±10%
of the control value (n=4 cells), which was completely abolished by
Rp-8-CPT-cAMPS (94±5% of control value; n=4 cells).
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Discussion |
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The findings in the present study indicate that the exogenous administration of the NO donor SNAP can induce opposing effects on myocyte contraction, in agreement with recent reports.8 9 Additional experiments using NOC5, an NO donor chemically distinct from SNAP, confirm these observations. Although the direction of the myocardial response clearly depended on the concentration of the NO donor used and on the level of intracellular cGMP reached, the principal novel finding of the present study is that a previously unrecognized GC/cGMP-independent mechanism involving the activation of AC is also critical in mediating this response.
In rat cardiac myocytes, high concentrations of SNAP (producing
14.7 µmol/L NO) induced large increases in cGMP and reduced
myocyte contraction, whereas low concentrations of SNAP (producing in
the range of 0.1 to 0.5 µmol/L NO) induced only moderate
increases in cGMP and enhanced myocyte contraction. Furthermore, these
contractile effects were not induced by the thiol byproduct of
SNAP, NAP, after NO release. Similarly, concentrations of NOC5
producing 0.5 µmol/L NO also enhanced myocyte contraction.
One can deduce that these contractile effects with SNAP and NOC5 are
authentic effects of NO, because they are completely reversible in the
presence of the NO scavengers oxyhemoglobin and carboxy-PTIO. More
importantly, our results show that whereas a cGMP/PKG-mediated
diminished sensitivity of the contractile proteins to
Ca2+ is largely responsible for the negative
inotropic effect at higher SNAP concentrations, the positive
contractile response observed with low concentrations of SNAP is
determined primarily by a cAMP/PKA-dependent, but GC/cGMP-independent,
increase in the Cai. Although recent reports have
addressed the wide range of actions of NO (and of NO donors) on
myocardial contractility,3 4 5 6 7 8 9 the
subcellular mechanisms involved in the opposing contractile responses
have not been completely elucidated.
In frog cardiac myocytes and in human atrial cells, cGMP has been shown to decrease Ca2+ influx through L-type channels by the stimulation of PDE type II (cAMP phosphodiesterase).38 39 In rat cardiac myocytes, however, cGMP has been shown to modulate L-type channel activity through a PKG-dependent pathway that does not involve changes in cAMP levels.40 41 Because Ca2+ entry through L-type Ca2+ channels is the principal trigger for Ca2+ release from the sarcoplasmic reticulum, the inactivation of L-type channels by NO/cGMP should have a significant effect on the amount of Ca2+ released to the myofilaments and therefore contribute to the negative inotropic response. This seems not to be an important mechanism in our model system, in which the administration of SNAP (100 µmol/L) produced a slowly evolving negative inotropic effect that was not associated with a reduction in the Cai. Furthermore, in steady-state tetanic contraction experiments, SNAP (100 µmol/L) significantly decreased cell shortening during the tetanus, despite the achievement of similar peak [Ca2+]i levels compared with controls. Taken together, these results indicate that the negative inotropic response to high concentrations of SNAP, in isolated rat cardiac myocytes, is mediated primarily by a reduction in the myofilament sensitivity to Ca2+ rather than by a reduced Ca2+ availability at the myofibrils.
In rat cardiac myocytes, administration of relatively high concentrations of the cGMP analog 8-bromo-cGMP (50 µmol/L) produced strikingly similar effects to those observed with high concentrations of SNAP (ie, a comparable negative inotropic response without any change of the Cai).21 Evidence for a cGMP-mediated reduction in myofilament responsiveness to Ca2+ has also been demonstrated in studies performed on skinned cardiac fibers.42 Previous reports also show that PKG activation induces a rightward shift of the tension/pCa relation in skinned single rat ventricular myocytes, compatible with a reduction in Ca2+ sensitivity of troponin C,43 and that PKG mediates phosphorylation of troponin I at the same site as that phosphorylated by protein kinase A, a mechanism known to reduce the affinity of troponin C for Ca2+.44 45 The precise underlying mechanism for the reduced myofilament responsiveness to Ca2+ induced by high concentrations of SNAP and increased cGMP was not directly addressed in our experiments. However, the ability of the PKG inhibitor KT 5823 to completely abolish the decrease in intact myocyte shortening observed during the tetanus in the presence of SNAP (100 µmol/L) (as it was also shown to do in this model in the presence of 8-bromo-cGMP21 ) provides strong support for the concept that SNAP-induced PKG activation results in reduced myofilament responsiveness to Ca2+.
Although other reports have indicated that positive contractile
responses are induced by low concentrations of NO donors and
cGMP,8 9 22 46 47 the subcellular mechanisms involved have
received relatively little attention. Some evidence suggests that the
positive contractile response induced by NO donors could be the result
of an elevation of the intracellular levels of cAMP (and PKA
activation) due to a cGMP-dependent inhibition of the PDE type III (ie,
cGMP-inhibited PDE).8 9 Furthermore, low concentrations of
cGMP (0.1 to 10 µmol/L) as well as of the NO donor SIN-1 (0.1 to
10 nmol/L) were found to have a stimulatory effect on L-type
Ca2+ current.15 48 It seems likely,
therefore, that the inhibition of cAMP degradation, mediated by the
inactivation of the PDE type III after low cGMP levels accumulate,
could result in the cAMP/PKA-dependent stimulation of transmembrane
Ca2+ influx, increasing the trigger for the
Ca2+-induced Ca2+ release
process, and producing a larger Cai and myocyte
contraction. Nevertheless, in the current experiments, SNAP (1
µmol/L) was still capable of eliciting a pronounced positive
contractile response (via increasing the Cai) in
the presence of a background of tonically inhibited PDEs (via IBMX
pretreatment; Figure 7). In this setting, it seems unlikely that
cGMP accumulation, per se, could mediate additional PDE (type III)
inhibition (and cAMP accumulation) beyond that tonically present,
owing to the presence of IBMX, and this leads to the notion that the
NO-mediated positive inotropy can occur, at least in part, via
PDE-independent mechanisms.
However, a decrease in PDE activity is only one of the two possible
major mechanisms capable of increasing cAMP in intact cells, the other
being the "upstream" activation of AC. Our results show that the
positive contractile response of SNAP (1 µmol/L) is indeed
associated with a significant elevation of both basal cAMP and cGMP.
Nevertheless, even when this basal cGMP increase was completely
abolished by the presence of the selective inhibitor of
soluble GC (ODQ) in the bathing solution (see Figure 5B), SNAP
(1 µmol/L) was still able to induce a positive inotropic effect
that was mediated by cAMP (Figures 5A and 9). In these
settings, cAMP must be increased by a mechanism other than
cGMP-mediated PDE inhibition, in all likelihood by a cGMP-independent
activation of AC.
To evaluate the possible activation of AC by the NO donor SNAP, we
determined AC activity in isolated sarcolemmal membranes from rat
heart. SNAP induced a concentration-dependent biphasic response of AC
activity, significantly increasing its activity at low doses (0.1 to
1 µmol/L) and either not changing (10 µmol/L) or
marginally decreasing the activity at high doses (100 µmol/L).
Consistent with our findings, recent studies provide evidence
suggesting that NO released from endothelial cells can
directly or indirectly activate AC in a cGMP-independent manner
in isolated perfused rat kidneys.49 From the present
experiments, we cannot ascertain whether NO activates AC
directly or through a G protein
(Gs/Gi)–dependent pathway.
Indeed, recent reports have demonstrated that NO can modulate G protein
function. Lander et al50 have demonstrated that treatment
of intact human peripheral blood mononuclear cells with NO
yielded membranes with enhanced GTPase activity, and that NO similarly
enhanced the GTPase activity of pure, recombinant
Gs
,
Gii, and
p21ras. Miyamoto et al51 found in
endothelial cells that NO can selectively inhibit G
proteins of the Gi and Gq
family but not those of the Gs family, and that
this modulation of G proteins could have a permissive action on the
Gs-AC pathway. Thus, investigation of this
question of the potential modulation of G protein signaling by NO in
cardiac myocytes should prove directly applicable to the issue of how
NO mediates the activation of AC and the positive inotropic effect.
The final remaining possibility to explain changes in AC activation in these experiments is that NO may modulate the activity of the ß-adrenergic receptor (ßAR) itself. It is noteworthy that the cAMP/PKA-mediated positive contractile effect of low concentrations of NO was associated neither with changes in the Cai duration nor a lusitropic effect. Although this lack of effect on contraction and Cai kinetics does not resemble the typical abbreviation of these parameters observed on ß1-adrenergic receptor (ß1AR) stimulation (which is also mediated by cAMP/PKA activation), on the other hand, it closely resembles the effects of another cAMP/PKA signaling cascade activated by the specific stimulation of ß2-adrenergic receptors (ß2ARs).52 Like the NO-induced positive inotropic effect, ß2AR stimulation produces a slowly evolving increase in contraction amplitude (as compared with ß1AR stimulation) without affecting the kinetics of contraction or the Cai.52 The differential regulation of cardiac excitation-contraction coupling by ß1AR and ß2AR stimulation has been attributed to a unique functional compartmentalization of cAMP.37 Thus, it is tempting to speculate that a simple and unifying explanation for all the experimental results at low NO concentrations involves the specific activation of ß2ARs, or perhaps of G proteins specifically coupled to them, but this will require experimental verification.
The nature of the regulation of protein function by NO is an area of
active investigation. Recent evidence suggests that thiols in proteins
can recognize both nitrosative and oxidative events, which in turn may
elicit distinct functional changes.27 28 29 For example, the
extent of reversible poly-S-nitrosylation of thiol groups
(R-S-NO) by NO was shown to correlate with the degree of activation of
the cardiac Ca2+ release channel (ryanodine
receptor), and this mode of posttranslational sulfhydryl modification
has been proposed as a general model of NO-mediated regulation of
protein function.27 29 These mechanisms could
plausibly operate at each of the effector proteins potentially involved
in the NO-stimulated production of cAMP in these experiments,
including the ß-ARs, G proteins, and AC, each of which has multiple
potential sites available for S-nitrosylation (ie, thiols of
the many cysteine residues). Indeed, posttranslational modification of
certain cysteines on these proteins has been shown to play an important
role in several hormone signal transduction pathways, including the
ßAR system. For example, labile thioesterification via palmitoylation
of a specific cysteine in the ß2ARs (and many
other G protein–coupled receptors), as well as
Gs, and the agonist-promoted dynamic turnover
of the binding of this moiety, has been shown to influence the ability
of these effectors to regulate the activation of
AC.53 54 Thus, competition for these thiol sites by
NO could exert significant regulatory effects on AC activation
independently of, or in cooperation with, the usual agonist induction
mechanisms.
Pinsky et al11 showed that endogenous cardiac
NO levels achieve dynamic, beat-to-beat oscillations on the
order of micromolar magnitude, and that this phenomenon is amplified by
ventricular loading, which could potentially serve an
autoregulatory function. Indeed, it has recently been shown that
intracardiac production and release of NO significantly
augments the Frank-Starling response.26 These
physiological NO levels in heart are thus
comparable to that achieved in the present experiments using 0.1 to
1 µmol/L SNAP or 1 µmol/L NOC5 (0.1 to 0.5
µmol/L NO) and associated with a positive inotropic effect. If the
rate of denitrosylation of R-S-NO is sufficiently slow (ie, on the time
scale of the cardiac cycle), then it is possible that these
physiological NO oscillations achieve a
functional integration or steady-state bound level, which in turn,
could proportionately regulate the function of the effector protein.
Such proportionate control of protein function via the degree of
protein S-nitrosylation has been demonstrated for the
cardiac ryanodine receptor, for example.29 It is thus
possible that the activation of AC demonstrated in these experiments at
levels of NO compatible with that achieved by the beating heart could
indeed serve a physiological autoregulatory role.
Notably, it has been shown that both myocardial cAMP55 56 57 58
and PKA55 activities oscillate during the cardiac cycle
(their peaks coinciding with mechanical systole), and this phenomenon
could thus be due, in part, to the NO-AC activation mechanism discussed
here. Still, a potential caveat should be raised insofar as the current
experiments were conducted at 25°C (and the contraction experiments
at 0.5-Hz electrical stimulation rate) and thus may not be directly
relevant to normal heart.
The exact nature for the biphasic activation pattern of AC by SNAP cannot be elucidated from our study. However, it seems reasonable to speculate that in isolated membranes, in which the NO buffering capacity of myoglobin59 60 is absent, NO released by high doses of SNAP would achieve substantially higher levels in the local environment of the membrane-bound AC than in intact cells (yielding an apparent leftward shift in the dose-response curve in isolated membranes compared with in vivo) and thus have nonspecific toxic (or inactivating) rather than activating effects on AC activity. Lander et al,50 when measuring GTPase activity induced by NO in isolated human peripheral blood mononuclear cell membranes, observed a similar biphasic response of enzyme activity (ie, activation with low concentrations of NO and inhibition with high concentrations of NO), attributing the inhibition that occurred at high doses to alteration of target proteins by nonspecific oxidation of thiols and tyrosine residues leading to enzyme inactivation.
In summary, the present results demonstrate that exogenously applied NO generated spontaneously by the donor SNAP, when given at high concentrations, can produce a negative inotropic effect that is mediated by a cGMP-dependent PKG activation resulting in the reduction in myofilament responsiveness to Ca2+ but not by decreased Ca2+ availability. Low concentrations of NO, on the contrary, evoke a positive inotropic effect determined primarily by a cAMP-dependent increase in the Cai by a novel mechanism whereby NO increases cAMP levels in intact rat ventricular myocytes by a cGMP-independent activation of AC.
|
Acknowledgments |
|---|
We acknowledge the expert technical assistance of Dr Xiaolin Wang, who performed the cAMP and cGMP assays.
|
Footnotes |
|---|
Preliminary findings previously presented in abstract form (Circulation. 1995;92[suppl S]:2412; J Mol Cell Cardiol. 1996;28:A126).
Received June 4, 1998; accepted February 22, 1999.
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References |
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F. Vandeput, S. L. Wolda, J. Krall, R. Hambleton, L. Uher, K. N. McCaw, P. B. Radwanski, V. Florio, and M. A. Movsesian Cyclic Nucleotide Phosphodiesterase PDE1C1 in Human Cardiac Myocytes J. Biol. Chem., November 9, 2007; 282(45): 32749 - 32757. [Abstract] [Full Text] [PDF]
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D. A. Kass, E. Takimoto, T. Nagayama, and H. C. Champion Phosphodiesterase regulation of nitric oxide signaling Cardiovasc Res, July 15, 2007; 75(2): 303 - 314. [Abstract] [Full Text] [PDF]
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M. Zaccolo and M. A. Movsesian cAMP and cGMP Signaling Cross-Talk: Role of Phosphodiesterases and Implications for Cardiac Pathophysiology Circ. Res., June 8, 2007; 100(11): 1569 - 1578. [Abstract] [Full Text] [PDF]
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E. Takimoto, D. Belardi, C. G. Tocchetti, S. Vahebi, G. Cormaci, E. A. Ketner, A. L. Moens, H. C. Champion, and D. A. Kass Compartmentalization of Cardiac {beta}-Adrenergic Inotropy Modulation by Phosphodiesterase Type 5 Circulation, April 24, 2007; 115(16): 2159 - 2167. [Abstract] [Full Text] [PDF]
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Y. Sun, O. A. Carretero, J. Xu, N.-E. Rhaleb, F. Wang, C. Lin, J. J. Yang, P. J. Pagano, and X.-P. Yang Lack of Inducible NO Synthase Reduces Oxidative Stress and Enhances Cardiac Response to Isoproterenol in Mice With Deoxycorticosterone Acetate-Salt Hypertension Hypertension, December 1, 2005; 46(6): 1355 - 1361. [Abstract] [Full Text] [PDF]
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R. Hambleton, J. Krall, E. Tikishvili, M. Honeggar, F. Ahmad, V. C. Manganiello, and M. A. Movsesian Isoforms of Cyclic Nucleotide Phosphodiesterase PDE3 and Their Contribution to cAMP Hydrolytic Activity in Subcellular Fractions of Human Myocardium J. Biol. Chem., November 25, 2005; 280(47): 39168 - 39174. [Abstract] [Full Text] [PDF]
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Q. Zhang, M. Lazar, B. Molino, R. Rodriguez, T. Davidov, J. Su, J. Tse, H. R. Weiss, and P. M. Scholz Reduction in interaction between cGMP and cAMP in dog ventricular myocytes with hypertrophic failure Am J Physiol Heart Circ Physiol, September 1, 2005; 289(3): H1251 - H1257. [Abstract] [Full Text] [PDF]
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E.J.F. Danson, Y.H. Zhang, C.E. Sears, A.R. Edwards, B. Casadei, and D.J. Paterson Disruption of inhibitory G-proteins mediates a reduction in atrial {beta}-adrenergic signaling by enhancing eNOS expression Cardiovasc Res, September 1, 2005; 67(4): 613 - 623. [Abstract] [Full Text] [PDF]
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J. Layland, R. J. Solaro, and A. M. Shah Regulation of cardiac contractile function by troponin I phosphorylation Cardiovasc Res, April 1, 2005; 66(1): 12 - 21. [Abstract] [Full Text] [PDF]
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A. F. Leite-Moreira and C. Bras-Silva Inotropic effects of ETB receptor stimulation and their modulation by endocardial endothelium, NO, and prostaglandins Am J Physiol Heart Circ Physiol, September 1, 2004; 287(3): H1194 - H1199. [Abstract] [Full Text] [PDF]
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S. Calaghan and E. White Activation of Na+-H+ exchange and stretch-activated channels underlies the slow inotropic response to stretch in myocytes and muscle from the rat heart J. Physiol., August 15, 2004; 559(1): 205 - 214. [Abstract] [Full Text] [PDF]
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Q. Qin, X.-M. Yang, L. Cui, S. D. Critz, M. V. Cohen, N. C. Browner, T. M. Lincoln, and J. M. Downey Exogenous NO triggers preconditioning via a cGMP- and mitoKATP-dependent mechanism Am J Physiol Heart Circ Physiol, August 1, 2004; 287(2): H712 - H718. [Abstract] [Full Text] [PDF]
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J. F. Wen, X. Cui, J. Y. Jin, S. M. Kim, S. Z. Kim, S. H. Kim, H. S. Lee, and K. W. Cho High and Low Gain Switches for Regulation of cAMP Efflux Concentration: Distinct Roles for Particulate GC- and Soluble GC-cGMP-PDE3 Signaling in Rabbit Atria Circ. Res., April 16, 2004; 94(7): 936 - 943. [Abstract] [Full Text] [PDF]
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R. Schulz, M. Kelm, and G. Heusch Nitric oxide in myocardial ischemia/reperfusion injury Cardiovasc Res, February 15, 2004; 61(3): 402 - 413. [Abstract] [Full Text] [PDF]
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D. A. Wink, K. M. Miranda, T. Katori, D. Mancardi, D. D. Thomas, L. Ridnour, M. G. Espey, M. Feelisch, C. A. Colton, J. M. Fukuto, et al. Orthogonal properties of the redox siblings nitroxyl and nitric oxide in the cardiovascular system: a novel redox paradigm Am J Physiol Heart Circ Physiol, December 1, 2003; 285(6): H2264 - H2276. [Abstract] [Full Text] [PDF]
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J. Su, S. Zhang, J. Tse, P. M. Scholz, and H. R. Weiss Alterations in nitric oxide-cGMP pathway in ventricular myocytes from obese leptin-deficient mice Am J Physiol Heart Circ Physiol, November 1, 2003; 285(5): H2111 - H2117. [Abstract] [Full Text] [PDF]
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T. Reffelmann and R. A. Kloner Effects of sildenafil on myocardial infarct size, microvascular function, and acute ischemic left ventricular dilation Cardiovasc Res, August 1, 2003; 59(2): 441 - 449. [Abstract] [Full Text] [PDF]
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T. Reffelmann and R. A. Kloner Therapeutic Potential of Phosphodiesterase 5 Inhibition for Cardiovascular Disease Circulation, July 15, 2003; 108(2): 239 - 244. [Full Text] [PDF]
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M. T. Ziolo and D. M. Bers The Real Estate of NOS Signaling: Location, Location, Location Circ. Res., June 27, 2003; 92(12): 1279 - 1281. [Full Text] [PDF]
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C. Badorff and S. Dimmeler NO Balance: Regulation of the Cytoskeleton in Congestive Heart Failure by Nitric Oxide Circulation, March 18, 2003; 107(10): 1348 - 1349. [Full Text] [PDF]
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T. Peng, X. Lu, M. Lei, and Q. Feng Endothelial Nitric-oxide Synthase Enhances Lipopolysaccharide-stimulated Tumor Necrosis Factor-alpha Expression via cAMP-mediated p38 MAPK Pathway in Cardiomyocytes J. Biol. Chem., February 28, 2003; 278(10): 8099 - 8105. [Abstract] [Full Text] [PDF]
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P B Massion and J-L Balligand Modulation of cardiac contraction, relaxation and rate by the endothelial nitric oxide synthase (eNOS): lessons from genetically modified mice J. Physiol., January 1, 2003; 546(1): 63 - 75. [Abstract] [Full Text] [PDF]
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R. Rubio and G. Ceballos Sole activation of three luminal adenosine receptor subtypes in different parts of coronary vasculature Am J Physiol Heart Circ Physiol, January 1, 2003; 284(1): H204 - H214. [Abstract] [Full Text] [PDF]
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J. Han, N. Kim, H. Joo, E. Kim, and Y. E. Earm ATP-sensitive K+ channel activation by nitric oxide and protein kinase G in rabbit ventricular myocytes Am J Physiol Heart Circ Physiol, October 1, 2002; 283(4): H1545 - H1554. [Abstract] [Full Text] [PDF]
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J. Layland, J.-M. Li, and A. M Shah Role of cyclic GMP-dependent protein kinase in the contractile response to exogenous nitric oxide in rat cardiac myocytes J. Physiol., April 15, 2002; 540(2): 457 - 467. [Abstract] [Full Text] [PDF]
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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,ACh in rat cardiac myocytes J. Physiol., April 15, 2002; 540(2): 411 - 424. [Abstract] [Full Text] [PDF]
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D. B. Sawyer and J. Loscalzo Myocardial Hibernation: Restorative or Preterminal Sleep? Circulation, April 2, 2002; 105(13): 1517 - 1519. [Full Text] [PDF]
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W. F. Saavedra, N. Paolocci, M. E. St. John, M. W. Skaf, G. C. Stewart, J.-S. Xie, R. W. Harrison, J. Zeichner, D. Mudrick, E. Marban, et al. Imbalance Between Xanthine Oxidase and Nitric Oxide Synthase Signaling Pathways Underlies Mechanoenergetic Uncoupling in the Failing Heart Circ. Res., February 22, 2002; 90(3): 297 - 304. [Abstract] [Full Text] [PDF]
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K. C. Wollert, B. Fiedler, S. Gambaryan, A. Smolenski, J. Heineke, E. Butt, C. Trautwein, S. M. Lohmann, and H. Drexler Gene Transfer of cGMP-Dependent Protein Kinase I Enhances the Antihypertrophic Effects of Nitric Oxide in Cardiomyocytes Hypertension, January 1, 2002; 39(1): 87 - 92. [Abstract] [Full Text] [PDF]
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M. T. Ziolo, H. Katoh, and D. M. Bers Positive and negative effects of nitric oxide on Ca2+ sparks: influence of beta -adrenergic stimulation Am J Physiol Heart Circ Physiol, December 1, 2001; 281(6): H2295 - H2303. [Abstract] [Full Text] [PDF]
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 H. SENZAKI, C. J. SMITH1, G. J. JUANG, T. ISODA, S. P. MAYER, A. OHLER, N. PAOLOCCI, G. F. TOMASELLI, J. M. HARE, and D. A. KASS Cardiac phosphodiesterase 5 (cGMP-specific) modulates {beta}-adrenergic signaling in vivo and is down-regulated in heart failure FASEB J, August 1, 2001; 15(10): 1718 - 1726. [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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R. D. Lasley, M. S. Jahania, and R. M. Mentzer Jr. Beneficial effects of adenosine A2a agonist CGS-21680 in infarcted and stunned porcine myocardium Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1660 - H1666. [Abstract] [Full Text] [PDF]
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S. J. Zieman, G. Gerstenblith, E. G. Lakatta, G. O. Rosas, K. Vandegaer, K. M. Ricker, and J. M. Hare Upregulation of the Nitric Oxide-cGMP Pathway in Aged Myocardium : Physiological Response to l-Arginine Circ. Res., January 19, 2001; 88(1): 97 - 102. [Abstract] [Full Text] [PDF]
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A. Lochner, E. Marais, S. Genade, and J. A. Moolman Nitric oxide: a trigger for classic preconditioning? Am J Physiol Heart Circ Physiol, December 1, 2000; 279(6): H2752 - H2765. [Abstract] [Full Text] [PDF]
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F. Chen Muscarinic regulation of cardiac L-type Ca2+ current Cardiovasc Res, December 1, 2000; 48(3): 362 - 364. [Full Text] [PDF]
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G. Klein, H. Drexler, and F. Schroder Protein kinase G reverses all isoproterenol induced changes of cardiac single L-type calcium channel gating Cardiovasc Res, December 1, 2000; 48(3): 367 - 374. [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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R. Ullrich, M. Scherrer-Crosbie, K. D. Bloch, F. Ichinose, H. Nakajima, M. H. Picard, W. M. Zapol, and Z. M. N. Quezado Congenital Deficiency of Nitric Oxide Synthase 2 Protects Against Endotoxin-Induced Myocardial Dysfunction in Mice Circulation, September 19, 2000; 102(12): 1440 - 1446. [Abstract] [Full Text] [PDF]
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J. M. Canty Jr. Nitric Oxide and Short-Term Hibernation : Friend or Foe? Circ. Res., July 21, 2000; 87(2): 85 - 87. [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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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. Han, N. Kim, E. Kim, W.-K. Ho, and Y. E Earm Modulation of ATP-sensitive Potassium Channels by cGMP-dependent Protein Kinase in Rabbit Ventricular Myocytes J. Biol. Chem., June 15, 2001; 276(25): 22140 - 22147. [Abstract] [Full Text] [PDF]
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J. Layland, J.-M. Li, and A. M Shah Role of cyclic GMP-dependent protein kinase in the contractile response to exogenous nitric oxide in rat cardiac myocytes J. Physiol., April 15, 2002; 540(2): 457 - 467. [Abstract] [Full Text] [PDF]
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J. W. Wegener, H. Nawrath, W. Wolfsgruber, S. Kuhbandner, C. Werner, F. Hofmann, and R. Feil cGMP-Dependent Protein Kinase I Mediates the Negative Inotropic Effect of cGMP in the Murine Myocardium Circ. Res., January 11, 2002; 90(1): 18 - 20. [Abstract] [Full Text] [PDF]
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W. F. Saavedra, N. Paolocci, M. E. St. John, M. W. Skaf, G. C. Stewart, J.-S. Xie, R. W. Harrison, J. Zeichner, D. Mudrick, E. Marban, et al. Imbalance Between Xanthine Oxidase and Nitric Oxide Synthase Signaling Pathways Underlies Mechanoenergetic Uncoupling in the Failing Heart Circ. Res., February 22, 2002; 90(3): 297 - 304. [Abstract] [Full Text] [PDF]
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G. U. Ahmmed, Y. Xu, P. Hong Dong, Z. Zhang, J. Eiserich, and N. Chiamvimonvat Nitric Oxide Modulates Cardiac Na+ Channel via Protein Kinase A and Protein Kinase G Circ. Res., November 23, 2001; 89(11): 1005 - 1013. [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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D. K. Kalra, X. Zhu, M. K. Ramchandani, G. Lawrie, M. J. Reardon, D. Lee-Jackson, W. L. Winters, N. Sivasubramanian, D. L. Mann, and W. A. Zoghbi Increased Myocardial Gene Expression of Tumor Necrosis Factor-{alpha} and Nitric Oxide Synthase-2: A Potential Mechanism for Depressed Myocardial Function in Hibernating Myocardium in Humans Circulation, April 2, 2002; 105(13): 1537 - 1540. [Abstract] [Full Text] [PDF]
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