1 Department of Biomedical Engineering, Duke University, Durham, North Carolina 27708; 3 Division of Cardiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15213; 4 Department of Physiology, University of North Carolina-Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599; and 2 Department of Chemistry, Oakland University, Rochester, Michigan 48309
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ABSTRACT |
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Nitric oxide (NO) has been implicated in endogenous control of
myocardial contractility. However, NO release has not yet been demonstrated in cardiac myocytes. Accordingly, endogenous NO production was measured with a porphyrinic microsensor positioned on the surface
of individual neonatal or adult rat ventricular myocytes (n > 6 neonatal and adult cells per
experiment). In beating neonatal myocytes, there was no detectable
spontaneous NO release with each contraction. However, norepinephrine
(NE; 0.25-1 µM) elicited transient NO release from beating
neonatal (149 ± 11 to 767 ± 83 nM NO) and noncontracting adult
(157 ± 13 to 791 ± 89 nM NO) cells. NO was released by
adrenergic agonists with the following rank order of potency:
isoproterenol
(1
2) > NE (
/
1) > dobutamine (
1)
epinephrine
(
/
1
2) > tertbutylene (
2); NO was
not released by phenylephrine (
). NE-evoked NO release was
reversibly blocked by
NG-monomethyl-L-arginine,
trifluoperazine, guanosine
5'-O-(2-thiodiphosphate), and
nifedipine but was enhanced by 3-isobutyl-1-methylxanthine (0.5 mM = 14.5 ± 1.6%) and BAY K 8644 (10 µM = 11.9 ± 1%). NO was
also released by A-23187 (10 µM = 884 ± 88 nM NO), guanosine 5'-O-(3-thiotriphosphate) (1 µM = 334 ± 56 nM
NO), and dibutyryl adenosine 3',5'-cyclic monophosphate
(10-100 µM = 35 ± 9 to 284 ± 49 nM NO) but not by ATP,
bradykinin, carbachol, 8-bromoguanosine 3',5'-cyclic
monophosphate, or shear stress. This first functional demonstration of
a constitutive NO synthase in cardiac myocytes suggests its regulation
by a
-adrenergic signaling pathway and may provide a novel mechanism
for the coronary artery vasodilatation and enhanced diastolic
relaxation observed with adrenergic stimulation.
norepinephrine; -adrenergic agonists; nitric oxide-selective
porphyrinic microsensor
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INTRODUCTION |
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THE PRESENCE OF a constitutive nitric oxide synthase (cNOS) in cardiac myocytes was first suggested by two studies investigating Ca2+-independent inducible NOS (iNOS) activity. One study noted negative inotropic effects within minutes of the application of inflammatory cytokines to isolated papillary muscles, before iNOS upregulation (7). The other study observed Ca2+-dependent conversion of L-[14C]arginine to L-[14C]citrulline in the cytosolic homogenates of myocytes (25). However, several contradictions arise when considering the presence of a cardiac cNOS. Because cholinergic stimulation (acetylcholine and carbachol) decreases myocardial contractility and nitric oxide (NO) has similarly been reported to decrease contractility (4, 7), one might predict that cholinergic stimulation would evoke NO release. On the other hand, adrenergic stimulation [norepinephrine (NE) and epinephrine] increases myocardial contractility by elevating cytoplasmic Ca2+ levels, which should also stimulate NO release. In addition, since cardiac myocytes undergo cyclic elevations in cytoplasmic Ca2+ as a prerequisite to contraction, beat-to-beat activation of a Ca2+-dependent cNOS might also be predicted, but beat-to-beat NO release might also inhibit adrenergically evoked increases in myocardial contractility.
Several studies, not directly measuring NO, have reported that cardiac
myocytes produce NO in response to acetylcholine (1, 3, 9). In one
study, this conclusion was based on experiments in open-chest dogs, in
which the vagus nerve innervating the heart was stimulated to release
acetylcholine (9). Because decreases in contractility due to vagal
stimulation could be opposed by systemic administration of
NG-monomethyl-L-arginine
(L-NMMA), a competitive
inhibitor of NO production, it was interpreted that acetylcholine was
evoking, and L-NMMA inhibiting,
NO production by cardiac myocytes. However, it is equally plausible
that the observed effects were due to acetylcholine-evoking and
L-NMMA-inhibiting NO release
from vascular endothelial cells in the heart. Acetylcholine-evoked NO
release from vascular endothelium is well established. In another
study, from isolated rat ventricular myocytes,
L-NMMA was reported to prevent
cholinergic agonists from opposing adrenergically mediated increases in
voltage-dependent Ca2+ currents
(3). However, similar studies in frog ventricular myocytes reported no
effects of L-NMMA on cholinergic
modulation of voltage-dependent
Ca2+ currents (16). Finally,
transgenic mice that overexpress
2-adrenergic receptors have
been shown to exhibit enhanced myocardial relaxation (24), similar to
the observed effects of NO on cardiac function (22). This raises the
possibility that cardiac myocytes may produce NO in response to
adrenergic agonists.
To test the hypothesis that cardiac myocytes contain a cNOS and to characterize its regulatory pathway, we used a highly selective porphyrinic microsensor to directly measure endogenous NO production by individual myocytes exposed to a variety of putative agonists, antagonists, and second messenger intermediates of NO release.
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METHODS |
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NO electrode preparation.
Nafion-coated porphyrinic microsensors (tip diameter,
5-15 µm; NO detection limit, 5 nM; response time, 1 ms) were
prepared, with modifications, as previously described (12). Carbon
strands (1-5 fibers, 5 µm diameter each; Amoco, Greenville, SC)
were inserted into glass capillary tubes (1 mm internal
diameter) pulled to a 100-µm opening at one end. Copper wire was connected to the carbon fiber, with electrically conductive epoxy at the blunt end, while the strand protruded at the pulled end.
The opening at the pulled end was sealed with molten wax, and the
protruding fiber was cut to the desired length (2 mm). The 2-mm-long
carbon fiber was coated with
tetrakis(3-methoxy-4-hydroxyphenyl)-nickel(II)porphyrin (TMHPPNi),
synthesized according to a procedure previously described (15).
Monomeric TMHPPNi was dissolved in 0.1 N NaOH and deposited, as a
polymeric film, on the carbon fiber using a multiple-potential scanning
cyclic voltameter (0.2 to +1.0 V, model 283 Potentiostat/Galvanostat; EG&G Instruments, Princeton Applied Research,
Princeton, NJ). Polymeric TMHPPNi catalyzes the oxidation of
NO to NO+. After the porphyrin
dried (2-3 h), the cation exchanger Nafion (Aldrich Chemical) was
applied by dipping the electrode into a 1% solution made up in
ethanol. The negative charges of the
functional group of Nafion
keeps
(nitrite) and
(nitrate) anions from gaining
access to the active porphyrin surface, preventing overestimation of the NO response. The Nafion-coated electrodes were characterized by
differential pulse voltametry to determine the redox potential of the
oxidation of NO to NO+.
Chronoamperometry, performed at a constant potential 50 mV more positive than the redox potential, was used to determine the NO concentration. High purity (>99.99%) NO standards (12) were prepared
daily to accurately calibrate the electrodes. The currents generated by
the oxidation of NO to NO+ at the
porphyrinic interface (0.5-1.5
nA/cm2 for 1 µM NO under static
conditions) were amplified and converted to voltages (model 283 Potentiostat/Galvanostat) and then were digitized for real-time viewing
on a monitor or stored on a computer hard disk for later retrieval and
analysis.
Isolation of neonatal and adult cardiac ventricular
myocytes. Neonatal myocytes were prepared from the
ventricles of 1- to 2-day-old halothane (1%) anesthetized rats as we
previously described (18). Noncontracting
Ca2+-tolerant adult myocytes were
prepared from Sprague-Dawley rats (250-300 g, either sex). After
deep anesthesia with pentobarbital sodium (40 mg/kg ip), the thoracic
cavity was opened and the heart was rapidly excised, cannulated at the
aorta, and retrogradely perfused (37°C; 120 mmHg) in a modified
Langendorff preparation. Hearts were first perfused for 5 min
with a solution containing (in mM) 144 NaCl, 5.4 KCl, 0.4 NaH2PO4,
10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 1 MgCl2, and 1.8 CaCl2, pH 7.4, and then for 5 min
with a Ca2+-free solution
additionally containing (in mM) 0.1 ethylene glycol-bis(-aminoethyl ether)-N,N,N',N'-tetraacetic
acid (EGTA), 5 creatine, and 10 taurine, and, finally, for 15 min with
a Ca2+-free solution without EGTA
but additionally containing (in mg/ml) 1.5 collagenase, 0.5 protease,
and 0.01 elastase. After perfusion, the atria and right ventricle were
removed and the remaining left ventricle was minced and incubated, with
stirring, in the collected enzyme mix additionally containing bovine
serum albumin (10 mg/ml). Aliquots (10 ml) of the stirring enzyme mix
containing suspended dissociated cells were centrifuged at 1,000 revolutions/min for 5 min. The supernatant was returned to the stirring
enzyme mix, and the pellet was resuspended in medium 199 (Sigma
Chemical). The cycle was repeated until the tissue was completely
dissociated. The cells in medium 199 were kept at 25°C for
immediate use.
Measurements of NO release from neonatal and adult cardiac ventricular myocytes. Neonatal myocytes were cultured on 10-mm-diameter collagen-coated glass coverslips; a suspension of adult myocytes was gently applied, dropwise, to collagen-coated glass coverslips and allowed to settle out and attach. The glass coverslip with cells was placed in a temperature-regulated bath (30 mm diameter, 2 ml vol, 37°C; Fig. 1). A glass disk forms the floor of the bath, which was mounted on the stage of an inverted microscope (Diaphot; Nikon). Perfusate [containing (in mM) 144 NaCl, 5.4 KCl, 1 MgCl2, 1.8 CaCl2, 10 glucose, and 10 phosphate; pH 7.4] entered the chamber from one end (1 ml/min) and drained out the other. Drugs were added to the bath along with the perfusate or were locally applied to a cell via a remote-controlled positive displacement nanoejector (Drummond Scientific, Broomall, PA). The nanoejector was mounted on a remote-controlled x-y-z micromanipulator with 1-µm resolution, which allows the glass tip of the ejector to be positioned upstream and proximal to the cell. Inert dye was added to the solution in the capillary tube of the ejector to visually guarantee that the drug was delivered on demand but did not otherwise diffuse out of the capillary tube. A three-electrode recording system was used, consisting of a working electrode (porphyrinic microsensor), saturated calomel reference electrode, and platinum counter electrode. The porphyrinic microsensor was mounted on a remote-controlled x-y-z ultramicromanipulator with 0.2-µm resolution, which allowed the tip of the microsensor to be positioned onto the surface of the cell.
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Immunohistochemical staining for iNOS protein and semiquantitative reverse transcriptase-polymerase chain reaction analysis for iNOS mRNA. Immunohistochemistry and reverse transcriptase-polymerase chain reaction (RT-PCR) were performed as we have previously described (18).
Statistical methods. Data are means ± SE for determinations in at least six different cells for microsensor recordings. Analysis of variance and the Student-Newman-Keuls test were used for multigroup comparisons. Values of P < 0.05 were considered statistically significant.
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RESULTS |
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Endogenous NO release was measured with a porphyrinic microsensor
placed directly on the surface of spontaneously beating neonatal or
noncontracting adult rat ventricular myocytes. The presence of a
functional Ca2+-dependent cNOS was
definitively demonstrated, for the first time, with the
Ca2+ ionophore A-23187 (Fig.
2A). NO
release began 1-2 s after the application of ionophore and
continued for 3-4 s. Transient release of NO was also elicited by
the adrenergic agonist NE (Fig. 2B). NO release began 100-200 ms after the application of NE and lasted for 1-6 s, depending on the concentration of agonist applied. The
participation of -adrenergic receptors in this response was indicated by the rank order of potency of selective adrenergic agonists: isoproterenol
(
1
2) > NE (
/
1) > dobutamine
(
1)
epinephrine
(
/
1
2) > tertbutylene (
2); NO was
not released by the
-agonist, phenylephrine (
) (data not shown).
NO release was blocked by the
-adrenergic antagonists, propranolol
(
1
2) > atenolol (
1) > butoxamine (
2) but not by the
-adrenergic antagonists, prazosin
(
1) or yohimbine
(
2) (data not shown).
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Verification that a measured NO response was from an individual myocyte was accomplished by raising the tip of the microsensor from the cell surface and applying NE (Fig. 2C). As the distance from the cell surface increased, there was a gradual decrease in the measured NO concentration. NO was undetectable beyond a distance of 30 µm (Fig. 2D). Mathematical modeling of the diffusion (12, 27) and breakdown (13) of NO indicated that the decline in concentration with distance was primarily due to degradation. NE increased the beating rate of neonatal cells (70 ± 6 to 128 ± 11 beats/min at 37°C; n = 7) and induced noncontracting adult cells to contract (98 ± 18 beats/min at 37°C; n = 8) as predicted (23). Positive chronotropy began within 15-30 s after the application of NE and lasted several minutes. However, only a single transient release of NO, beginning in 100-200 ms and lasting 1-6 s, followed each application of NE rather than a beat-to-beat release. Repetitive applications of agonist caused repetitive transient releases of NO.
Endothelial and neuronal cells possess cNOS, which are inhibited by L-NMMA (8) and are Ca2+/calmodulin dependent (5, 6). Cardiac cNOS was similarly inhibited by L-NMMA (Fig. 3A) and the calmodulin antagonist trifluoperazine (Fig. 3B). Evidence supporting the dependence of cardiac cNOS on extracellular Ca2+ was suggested by the inhibition of agonist-induced NO release with the L-type Ca2+ channel blockers, verapamil (Fig. 3C) and nifedipine (data not shown). Further evidence for a dependence on extracellular Ca2+ was provided by the enhancement of NE-evoked NO release with the L-type Ca2+ channel activator, BAY K 8644 (Fig. 3D).
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The release of NO by the nonhydrolyzable GTP analog, guanosine
5'-O-(3-thiotriphosphate) (GTPS), and blockade by
the nonhydrolyzable GDP analog, guanosine
5'-O-(2-thiodiphosphate)
(GDP
S), suggest that cardiac cNOS is regulated by a G
protein-dependent mechanism (Fig.
4A). The
regulation of cardiac cNOS by a adenosine 3',5'-cyclic monophosphate (cAMP)-dependent mechanism (10, 14) was also suggested by
NO release with the membrane-permeant cAMP analog dibutyryl adenosine
3',5'-cyclic monophosphate (DBcAMP; Fig.
4B). NO release due to NE was
enhanced by 3-isobutyl-1-methylxanthine (IBMX), which increases
intracellular cAMP and guanosine 3',5'-cyclic monophosphate
(cGMP) levels by inhibiting phosphodiesterases (Fig. 4C). The release of NO was not
induced or enhanced by 8-bromoguanosine 3',5'-cyclic
monophosphate (1 mM; n = 7). Thus cGMP
was not involved and IBMX-enhanced NE evoked NO release by prolonging
the half-life of cAMP. In sharp contrast to endothelial cells (12), NO
release was not elicited or enhanced by shear stress (0.2-10
dyn/cm2;
n = 6), ATP (10 µM;
n = 6), bradykinin (10 µM;
n = 7), or the cholinergic agonist
carbachol (10 µM; n = 7).
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iNOS has previously been reported in cardiac myocytes after exposure to
inflammatory cytokines (2, 18, 25). The absence of an inducible enzyme
in our cells was confirmed by immunohistochemical staining that was
negative for iNOS (Fig.
5A). In
contrast, cells treated with the proinflamatory cytokine,
interleukin-1 (IL-1), stained positive for iNOS protein (Fig.
5B) as previously reported (18). The
absence of iNOS mRNA was also confirmed by RT-PCR. Only IL-1-treated
cells exhibited an RT-PCR iNOS product (Fig. 5C).
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DISCUSSION |
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Pharmacological and molecular studies have suggested the existence of a cNOS in cardiac myocytes (1, 7, 25). However, the present study definitively demonstrates, for the first time, the presence of a functional cNOS in both neonatal and adult cardiac ventricular myocytes. Cardiac cNOS shares inhibition by L-NMMA and Ca2+/calmodulin dependence with both endothelial and neuronal cNOS. However, cardiac cells differ from endothelial cells by releasing NO in response to adrenergic rather than cholinergic agonists. Reports of cholinergic-mediated NO release from cardiac myocytes are controversial and based on studies where NO was not measured. In vivo studies involved blocking, with NOS inhibitors, the decreases in myocardial contractility that resulted from vagal stimulation (9). However, the released NO being blocked may have come from vascular endothelial cells that are known to produce NO in response to cholinergic agonists. In vitro studies involved opposing, with NOS inhibitors, cholinergic inhibition of adrenergically evoked increases in voltage-dependent Ca2+ currents (3). However, although these findings are interesting, other studies have found no effects of NOS inhibitors on cholinergic modulation of voltage-dependent Ca2+ currents (16).
-Adrenergic receptor regulation of cNOS was demonstrated by the
release of NO with selective
-adrenergic (isoproterenol, dobutamine,
and tertbutylene) but not selective
-adrenergic (phenylephrine) agonists. Because GTP
S directly evoked NO release and GDP
S
blocked adrenergically mediated NO release, a G protein-dependent
pathway is indicated in myocyte NO release (Fig.
4A). In addition, the responses of
myocytes to DBcAMP (Fig. 4B) and the
phosphodiesterase inhibitor IBMX (Fig.
4C) suggest that a cAMP-dependent
adrenergic signaling pathway is also present (10, 19). The enhancement of NE-evoked NO release by BAY K 8644 (Fig.
3D) and the blockade of release with
verapamil (Fig. 3C) further suggest
that these pathways are operating through L-type
Ca2+ channels.
It is generally accepted that -adrenergic receptors are coupled, via
G proteins, to adenylyl cyclase. The formation of cAMP and the effects
that it brings about through the phosphorylation of proteins typically
take from seconds to tens of seconds to develop. An example of this
time lag is the 15-30 s required for increases in chronotropy and
inotropy to develop in beating neonatal myocytes after the addition of
-adrenergic agonists. However, the onset of NE-evoked NO release
began within 100-200 ms of the addition of agonist, suggesting the
presence of a "fast"
-adrenergic/G protein pathway where G
proteins may be directly coupled to L-type Ca2+ channels (14, 28). If a fast
G protein-dependent pathway is responsible for NO release, it is
interesting to speculate that the "slow" cAMP-dependent pathway
is responsible for modulation of NO levels. Finally, NO was not
spontaneously released beat-to-beat from contracting cells. This
suggests that myocyte cNOS is compartmentalized and isolated from the
transient elevations in intracellular
Ca2+ responsible for
excitation-contraction coupling.
NO serves a paracrine function in a variety of cell types (17, 26). For example, NO released by endothelial cells acts on underlying smooth muscle cells to induce vasodilatation (11, 20, 21). Similarly, NO released from cardiac myocytes may be responsible for dilation of adjacent coronary resistance vessels. The resultant increase in coronary perfusion is necessary to support adrenergically induced increases in myocardial contractility. It is interesting to speculate on an autocrine role for this free radical in cardiac myocytes. NO produced by myocytes may enhance myocardial relaxation and regulate contractility as well as coronary perfusion. Consequently, deficiencies in cardiac myocyte cNOS and NO production may play a role in such diverse cardiac syndromes as coronary artery vasospasm and myocardial diastolic dysfunction.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute National Research Service Award Grant 1F32-HL-09072 and Grant-in-Aid NC-95-GB-08 from the American Heart Association, North Carolina Affiliate (A. J. Kanai); by Grant RO1-HL-53372 (M. S. Finkel); and by a grant from the Biotechnology Research Program of Oakland University (T. Malinski).
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FOOTNOTES |
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Address for reprint requests: A. J. Kanai, Box 90281, Dept. of Biomedical Engineering, Duke Univ., Durham, NC 27708.
Received 21 August 1996; accepted in final form 15 July 1997.
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