© 1996 American Heart Association, Inc.
Articles |
Nitric Oxide Synthase (NOS3)–Mediated Cholinergic Modulation of Ca2+ Current in Adult Rabbit Atrioventricular Nodal Cells
From the Cardiovascular Division, Department of Medicine (X.H., J.-L.B., R.A.K., T.W.S.) and Department of Pathology (L.K.), Brigham and Women's Hospital, Harvard Medical School, and Harvard School of Public Health, Boston, Mass.
Correspondence to Dr X. Han, Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St, Boston, MA 02115. E-mail xhan@bics.bwh.harvard.edu.
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Abstract |
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Abstract We examined the role of endogenous NO in the autonomic regulation of atrioventricular (AV) nodal function by studying spontaneous action potentials (SAPs) and L-type Ca2+ current (ICa-L) in isolated single AV nodal cells from adult rabbit hearts. Both the perforated and the membrane-ruptured patch-clamp techniques in the whole-cell configuration were used under conditions known to alter NO production. Three NO donors, 3-morpholinosydnonimine (SIN-1, 0.1 mmol/L), S-nitroso-acetylcysteine (0.1 mmol/L), and sodium nitroprusside (0.1 mmol/L), suppressed the ß-adrenergic agonist isoproterenol (ISO, 1 µmol/L)–stimulated increase in ICa-L. SIN-1 also decreased the frequency and amplitude of SAPs. In cells in which ICa-L had been previously attenuated by the muscarinic agonist carbamylcholine (CCh, 1 µmol/L), SIN-1 had no additive effect. CCh activated an acetylcholine-sensitive outward K+ current (IK(ACh)) in AV nodal cells, in addition to the ICa-L inhibition. Intracellular dialysis with the NO synthase inhibitor N-monomethyl-L-arginine (L-NMMA, 0.5 mmol/L) blocked CCh-induced, but not SIN-1–induced, ICa-L attenuation. However, intracellular dialysis with methylene blue (20 µmol/L), which inhibits NO-mediated activation of guanylyl cyclase and cGMP production, blocked the effects of both CCh and SIN-1 on ICa-L. In these cells, neither L-NMMA nor methylene blue affected the CCh-activated IK(ACh). Direct application of cGMP (10 µmol/L) via internal dialysis significantly inhibited ISO-stimulated ICa-L. In AV nodal cells internally perfused with either a nonhydrolyzable cAMP analogue, 8-Br-cAMP (0.5 mmol/L), or a high concentration of cAMP (0.5 mmol/L), CCh did not inhibit ICa-L but still activated IK(ACh). CCh-induced ICa-L attenuation could be abolished or quickly reversed by the nonselective phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (20 µmol/L). However, CCh still significantly suppressed ISO-stimulated ICa-L after the cGMP-inhibited PDE isozyme (PDE3) had been selectively inhibited by milrinone (5 µmol/L). Immunohistochemical staining identified the presence of the endothelial constitutive NO synthase (ecNOS or NOS3) in both single AV nodal cells in vitro and in cryostat sections of AV nodal tissue in situ. These results demonstrate that endogenous NO is involved in the muscarinic cholinergic attenuation of ICa-L in AV nodal cells; the mechanism likely involves the cGMP-stimulated PDE.
Key Words: nitric oxide • atrioventricular node • whole-cell patch clamp • isoproterenol • carbamylcholine
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Introduction |
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Nitric oxide is an important second messenger in the actions of some neurotransmitters in the cardiovascular and central nervous systems.1 2 3 NO is known to modulate cardiac contractility both in vitro4 5 and in vivo.6 Under pathological conditions, increased production of NO by cardiac muscle cells following inflammatory cytokine-mediated induction of NO synthase II (inducible NO synthase) causes a marked decrease in myocyte contractile responsiveness to ß-adrenergic stimulation.7 8 In the primary pacemaker region of the heart, the sinoatrial node, NO acts as an important mediator for the autonomic regulation of heart rate.9 10 11 Both the chronotropic and inotropic effects of NO appear to be mediated at least in part by an inhibition of ICa-L in the heart; the cellular signaling pathway involves activation of guanylyl cyclase, elevation of cGMP, and stimulation of a cAMP-specific PDE.10
The AV node has long been known to play specific roles in the electrical activity of the heart.12 13 The AV node constitutes a key component of the only region where electrical excitation normally passes from atria to ventricles.14 A large fraction of the delay between atrial and ventricular activation occurs at the AV node. This favors ventricular filling as a result of atrial contraction and thereby enhances the pumping function of the heart. The relatively long refractory period of the AV node prevents the ventricles from responding to an excessive atrial stimulus rate that otherwise could sustain life-threatening arrhythmias. Both the conduction delay and the refractory period that arise from the intrinsic properties of the AV node are modulated by the autonomic nervous system.12 13 15 16
Recently, we have successfully isolated viable, spontaneously active AV nodal cells from the adult rabbit heart9 10 11 17 18 and have characterized some of the ionic currents that are responsible for generating and conducting the AV nodal action potentials. In the present study, we addressed the following questions: (1) Do AV nodal cells contain NOS3? (2) What is the physiological role of NO in the AV node? (3) What are the subcellular mechanisms by which NO exerts its effects on AV nodal function? Our experiments, performed on freshly isolated single AV nodal cells under both nystatin-perforated and membrane-ruptured patch-clamp conditions, demonstrate that endogenous NO plays an important role in the muscarinic cholinergic inhibition of AV nodal SAPs and ICa-L. The NO-dependent intracellular signaling pathway may involve a cGMP-stimulated cAMP PDE.
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Materials and Methods |
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Cell Isolation
The technique used for isolation of single cells was as described previously.9 10 11 17 18 In brief, heparinized rabbits weighing 1 to 3 kg were anesthetized with pentobarbital and then killed by a blow to the neck. Hearts were quickly excised and Langendorff-perfused at 37°C with (1) bicarbonate-buffered Tyrode's solution for 3 minutes to wash out the blood, (2) Ca2+-free Tyrode's solution for 8 minutes, and (3) enzyme (12.5 U/mL collagenase, Yakult)–containing Ca2+-free Tyrode's solution for a further 10 minutes. Small segments of the AV node were excised from the intact hearts, and the surrounding tissues were removed under a dissecting microscope. The final preparations (
3x4 mm) were cut into four pieces and stirred
at 37°C in Ca2+-free, HEPES-buffered Tyrode's solution
containing 500 U/mL collagenase (type I, Sigma) and 0.1%
bovine serum albumin (fraction V, Sigma). Cells were collected,
centrifuged at 140g for 5 minutes, and stored at
4°C in modified KB solution19 (see below).
Solutions
The bicarbonate-buffered Tyrode's solution used for
Langendorff perfusion of the whole heart contained (mmol/L) NaCl
121, KCl 5.0, sodium acetate 2.8, MgCl2 1.0,
Na2HPO4 1.0, NaHCO3 24, glucose
5.5, and CaCl2 0.5. This solution was equilibrated with
95% O2/5% CO2 (pH 7.4). The KB
solution used for storing the cells contained (mmol/L) potassium
glutamate 90, potassium oxalate 10, KCl 25,
KH2PO4 10, NaOH 6, MgSO4 1.0,
taurine 20, HEPES 5, and glucose 10 (pH was adjusted to 7.2 with KOH).
The HEPES-buffered Tyrode's solution in which all experiments were
carried out contained (mmol/L) NaCl 145, KCl 5.4, MgCl2
1.0, Na2HPO4 1.0, HEPES 5.0, CaCl2
1.8, and glucose 10 (pH was adjusted to 7.4 with NaOH).
Recording Methods and Data Acquisition
In most experiments, the nystatin (0.3 mg/mL)–perforated patch
technique20 21 in the whole-cell
configuration22 was used to current- and voltage-clamp
the cells. The pipette solution contained (mmol/L) KCl 140, NaCl 6,
MgCl2 1, and HEPES 5 (pH was adjusted to 7.2 with KOH). DC
resistance was 1 to 3 M
. In some experiments using SNAC as the NO
donor, the gramicidin (0.3 mg/mL)–perforated recording
technique21 was used. Nystatin and gramicidin were first
dissolved in dimethyl sulfoxide and then added to the pipette solution.
There was no noticeable difference between the recordings made
with nystatin and gramicidin, although theoretically, a small junction
potential (<-8.8 mV) that is due to the Donnan-like
diffusion of Cl- may be developed with the
nystatin-perforated patches.20 In other experiments,
the conventional suction microelectrode ruptured-patch
whole-cell method was used. In these experiments, the pipette
solution included (mmol/L) potassium aspartate 95, KCl 30, HEPES 5,
disodium phosphocreatine 3, GTP (sodium) 0.1, K2ATP 3,
MgCl2 1, EGTA 10, and CaCl2 1 (pCa was in the
range of 7.4 to 7.5; pH was 7.2, adjusted with KOH). A liquid junction
potential of -10 mV was corrected electronically. The
electrode resistance was between 2 and 4 M. All recordings
were made at 32.5°C. Membrane currents were digitized at 2.5 KHz. The
volume of the recording chamber was 0.5 mL, and a constant flow
rate of 1.5 mL/min was used to superfuse the cells.
ICa-L was activated by
depolarizing to 0 mV for 150 to 200 milliseconds from a holding
potential of -40 mV and measured as the difference between the
holding and the peak inward current. No significant inactivation of
ICa-L was observed at a holding
potential of -40 mV over the time course of our experiments.
Because of its inward-rectifying property, IK(ACh)
changes little from -40 to 0 mV (and more positive potentials).
Activation of IK(ACh) shifts the holding current (at
-40 mV) in the outward (positive) direction. This shift also
occurs at 0 mV, at which peak inward Ca2+ current is
activated. Therefore, there is no net change in the difference
between the holding current and the peak
ICa-L. Since full activation of
ICa-L in the whole-cell mode
takes only several milliseconds (ie, the time to peak inward current),
any desensitization of IK(ACh) during this transient time
should be of negligible significance and will not affect the
measurement of ICa-L. Data were
discarded from experiments in which rundown of
ICa-L was >10%. Rundown was
retarded in our dialysis experiments by including sodium creatine and
ATP in the internal solution and by prestimulation
(phosphorylation) with the ß-adrenergic agonist
or cAMP analogues. Transient outward current and inward rectifier
K+ current were not detected in these AV nodal cells, which
facilitated the measurement of
ICa-L with our protocol.
Immunohistochemical Analysis of NOS3 in AV Nodal
Cells
Immunohistochemical analysis of NOS3 staining in AV
nodal cells was performed as described previously in adult rat
ventricular myocytes.5 Cryostat sections of
small AV nodal samples and freshly isolated single AV nodal cells were
fixed in buffered 2% paraformaldehyde for 5 minutes,
followed by 10 minutes in 100% methanol. Single AV nodal cells were
identified and documented to have characteristic SAPs under
nystatin-perforated recording conditions. After rinsing,
immunostaining was performed by sequential application
of primary antibody (5 µg/mL, mouse anti-human NOS3, Transduction
Laboratories), goat anti-mouse IgG (1/50, Steinberger Monoclonals,
Inc), and mouse peroxidase–antiperoxidase complex (1/100,
Steinberger Monoclonals, Inc), followed by labeling with the chromogen
diaminobenzidine (Sigma) and H2O2. The slides
were washed with water, counterstained with hematoxylin, dehydrated,
and mounted for light microscopy. Negative controls were treated the
same way, except a nonspecific mouse myeloma IgG (Sigma) was used as
the primary antibody.
Drugs and Chemicals
ISO, CCh, L-arginine, nystatin, sodium
nitroprusside, dimethyl sulfoxide, methylene blue, IBMX, and
8-Br-cGMP were purchased from Sigma Chemical Co. SIN-1 was obtained
from Molecular Probes, Inc. L-NAME was purchased from Bachem, Inc.
L-NMMA was obtained from Calbiochem. SNAC was made by mixing (1:1)
sodium nitrite and acetylcysteine at pH 7.4 (with NaOH).
Statistics
Statistical significance was evaluated by Student's unpaired
t test, where appropriate. Means with values of
P<.05 were considered to be significantly
different.
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Results |
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Immunohistochemical Identification of NOS3
Three isoforms of NO synthase have been identified to date, and our recent work has demonstrated the presence of NOS3 in ventricular myocytes from rat hearts.5 It is important to clarify whether specialized conduction tissue, such as AV nodal cells, also contain NOS3, because the neuronal isoform has been found as well in intracardiac ganglionic cells and in nerve endings innervating the AV node.23 24 As shown in Fig 1
, both single cells isolated from the
central AV nodal region (panels a and b) and cryostat sections of AV
node (panel d) show prominent diffuse cytoplasmic staining of NOS3,
denoted by the bright brown color, which is in clear contrast to the
background. No staining was detectable above background when the
primary antibody was omitted or nonspecific mouse myeloma IgG was
applied (panels c and e). In cryostat tissue sections (panels d and e),
which can be divided into three layers, cells at the endocardial
surface (highlighted by an arrow in panel d) covering the transitional
(letter T in panel d) and the central AV nodal regions (letter C in
panel d) also showed prominent positive staining of NOS3 (panel d),
which supports previous studies demonstrating that significant NOS3
activity is present in the endocardium.25 26 27
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Involvement of NO in Autonomic Regulation of SAPs and
ICa-L
With the nystatin-perforated patch under current clamp, all AV
nodal cells selected for the present study were beating
spontaneously and had other
electrophysiological characteristics
similar to those reported previously.16 17 18 SIN-1 (0.1
mmol/L), which releases NO in solution, significantly
decreased the frequency of SAPs after it had been increased by the
ß-adrenergic agonist ISO (1 µmol/L). Fig 2 shows
the effects of SIN-1. Traces of SAPs from panels A through E were
recorded consecutively under the control condition, ISO
stimulation, SIN-1 in the presence of ISO, washout of SIN-1, and
washout of ISO. Exposure to SIN-1 resulted in a reversible
decrease in the frequency of SAPs (compare panels C and D with panel
B). In six cells, the frequency of SAPs was 128±12
min-1 in the control condition, 164±23
min-1 in the presence of ISO, and 123±18
min-1 in the presence of ISO plus SIN-1.
SIN-1 also caused a small but significant decrease in the amplitude of
SAPs (4.5±0.5 mV) with little effect on the maximum
diastolic potential. At concentrations ranging from 1
nmol/L to 100 µmol/L, SIN-1 (n=2) did not affect the basal SAP
frequency.
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ICa-L is essential in generating
and maintaining AV nodal action potentials. The SIN-1–induced decrease
in the SAP frequency might be related to the inhibition of
ISO-stimulated ICa-L. This was
tested in the experiments shown in Fig 3, panels A
through C. On the top (panel A) is the time course of changes of
ICa-L (filled circles) and holding
current (open circles) in response to applications of ISO and SIN-1.
Panel B illustrates the original current traces recorded at the
time points denoted by the letters a through c in panel A. The averaged
changes in the peak ICa-L in
response to ISO and SIN-1 are plotted in panel C (n=5). ISO
significantly increased
ICa-L, which was
subsequently inhibited by exposure to the NO donor SIN-1. The holding
current remained unchanged in the presence of both agents. Similar
attenuation of ISO-stimulated ICa-L
was observed also with SNAC (n=7, panels D through F) and sodium
nitroprusside (0.1 mmol/L, n=3; data not shown). Neither SIN-1 (n=3)
nor SNAC (n=3) affected basal
ICa-L. In addition, 3 hours after
dissolving SIN-1 in solution, the same concentration of SIN-1 had no
effect on ISO-stimulated ICa-L
(n=2). Thus, these results in AV nodal cells confirm
our9 10 11 and other28 29 30 reports that exogenous
NO inhibits ISO-stimulated ICa-L in
the heart.
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) and holding current (
) in response to applications
of individual agents (ie, ISO, SIN-1, etc) are shown on the top, and
the original current traces recorded at the time points denoted by
the letters a through d in the time-course plot are illustrated on
the bottom. The horizontal lines in the time-course plot denote the
time during which each agent was present. In this and the
experimental data shown in Fig 4
Muscarinic cholinergic receptor activation is known to release
NO,1 2 3 and the presence of NOS3 in AV nodal cells
indicates that endogenous NO may participate in the
cholinergic regulation of ICa-L.
Experiments (n=3) illustrated in Fig 4 were designed to
test this possibility. The time course of changes in
ICa-L and holding current in
response to individual drug applications is shown in panel A, and
the current traces shown in panel B correspond to the time points
marked in panel A. The muscarinic agonist CCh (1 µmol/L) markedly
reduced (trace c) the ISO-stimulated
ICa-L (trace b), in addition to
activating a K+ current (IK(ACh)) seen as an
outward shift in the holding current. The mean values of peak
ICa-L in response to each test
agent application are plotted in panel C. The inhibitory
effect of SIN-1 on ICa-L was
preempted by CCh (trace d) at a time when the maximum reduction of
ICa-L was seen after CCh
application (trace c). These findings suggest that muscarinic receptor
activation and NO share a common biochemical pathway leading to the
inhibition of ISO-stimulated
ICa-L.
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If CCh-induced ICa-L inhibition
is mediated by endogenous NO, its effect should be blunted
or abolished by agents known to inhibit NO synthase. This was tested in
the experiments shown in Fig 5. In these experiments,
the membrane of the AV nodal cells was ruptured by gentle suction, and
the NO synthase inhibitor L-NMMA (0.5 mmol/L) was either
not included (panels A through C) or dialyzed into the cells (panels D
through F) through a pipette. The changes in
ICa-L (filled circles) and holding
current (open circles) in response to ISO and CCh are shown in panels A
and D, and the original current traces corresponding to the time points
a through c are illustrated in panels B and E. The percent changes in
the mean values of peak ICa-L
measured in the absence (n=5) and presence (n=5) of L-NMMA are plotted
in panels C and F, respectively. CCh significantly suppressed
ICa-L in addition to activating
IK(ACh) (panels A through C). However, when NOS was
inhibited by L-NMMA, there was little decrease in the ISO-stimulated
ICa-L by CCh, which only
activated IK(ACh) (panels D through F). Thus, these
experiments strongly suggest that the muscarinic cholinergic inhibition
of AV nodal cell ICa-L is not
simply due to the internal dialysis but, rather, may be mediated by an
endogenous NOS.
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Involvement of cGMP and cGMP-Stimulated cAMP PDE
In many tissues, NO activates guanylyl cyclase(s), which
then generates cGMP. Therefore, we attempted to inhibit the action of
CCh and NO on ICa-L in AV nodal cells by applying methylene
blue (20 µmol/L), a compound known to block NO-induced activation of
guanylyl cyclase.2 5 Two series of experiments were
performed, and the results are shown in Fig 6. The first
series of experiments (n=4) were performed with the
nystatin–perforated patch method. Both SIN-1 (0.1 mmol/L) and
methylene blue were applied extracellularly. As seen in panels A
through C, the inhibitory effect of SIN-1 on ISO-stimulated
ICa-L was blocked by methylene
blue. The peak ICa-L after SIN-1
application was 102±7% of the magnitude in ISO and methylene blue. In
the second series of experiments (panels D through F), the
membrane-ruptured whole-cell recording method was used
to allow intracellular dialysis with methylene blue. With this
protocol, in five cells, CCh significantly activated
IK(ACh) but failed to attenuate ICa-L. In the
presence of CCh, the mean peak
ICa-L was 94±6% of that in the
presence of ISO (panel F). The muscarinic receptor
antagonist atropine (1 µmol/L) quickly reversed the CCh
activation of IK(ACh) without affecting ISO-stimulated
ICa-L (panel D). Thus, these
experiments suggest that the inhibitory effects of CCh and
NO may be mediated through activation of guanylyl cyclase and elevation
of intracellular cGMP.
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Methylene blue may interfere with all thiol and reactive metal centers
in the cells. To confirm that the blockade of SIN-1- and
CCh-induced ICa-L inhibition is
due to methylene blue inhibition of guanylyl cyclase and reduction of
cGMP, we tested the effect of internal dialysis of cGMP (10 µmol/L)
on the ISO-stimulated ICa-L,
and the results are shown in Fig 7. In all of these
experiments, single AV nodal cells were preperfused with Tyrode's
solution containing 1 µmol/L ISO for 5 minutes before starting
internal dialysis. Beat-by-beat decrease in the ISO-stimulated
ICa-L was seen immediately after
dialyzing cGMP into the cell (panels A and B). This decrease usually
plateaued in 3 to 8 minutes; after which, the amplitude of
ICa-L remained relatively stable.
In eight cells, the maximum decrease after 3- to 8-minute dialysis was
49.5±4.5% (mean±SD), assuming that the value of
ICa-L obtained immediately after
dialysis is 100% (panel C).
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The following experiments were aimed at investigating the possible
biochemical events resulting from cGMP elevation and leading to the
ICa-L reduction. In both frog
myocardium and rabbit sinoatrial node, cGMP
stimulates a PDE that augments cAMP
breakdown,10 31 32 33 thereby attenuating the ISO-stimulated
ICa-L. Cyclic
nucleotide analogues that cannot be efficiently hydrolyzed
by PDE provide a useful experimental approach for evaluating the
possible involvement of PDE in the observed effects of acetylcholine
and NO. Two series of experiments were performed using the conventional
membrane-ruptured method to allow internal dialysis of the cyclic
nucleotides. In the first (n=5), a membrane-permeant
analogue of cAMP, 8-Br-cAMP, which directly activates
protein kinase A and is resistant to degradation by PDE, was
tested, and the results are shown in Fig 8A. The time
course of changes in ICa-L and
holding current are plotted on the top, and the original current traces
corresponding to the letters a through c are plotted on the bottom of
the figure. An increase in ICa-L
was seen immediately after the onset of dialysis with 8-Br-cAMP
(top) and gradually plateaued over 3 to 4 minutes, whereas the holding
current remained unchanged (trace b). In the presence of internal
8-Br-cAMP, CCh had no attenuating effect on
ICa-L but still significantly
activated IK(ACh) (trace c). Similar results were
obtained in a second series of experiments (n=4), when 0.5 mmol/L cAMP
was dialyzed into the cells (Fig 8B). Although cAMP is the natural
substrate for PDE, a constant supply of such a high concentration
should render its degradation insignificant within the time frame of
the present study. In a total of nine cells dialyzed with cAMP and
8-Br-cAMP, the mean increase in peak
ICa-L over control was 119±34% in
the absence and 110±22% in the presence of CCh. The outward current,
IK(ACh), activated by CCh was present in
all of our experiments (ie, ISO, 8-Br-cAMP, and cAMP), suggesting
that its activation is independent of the presence of these cyclic
nucleotides.
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That a cGMP-stimulated PDE mediates the inhibitory effects
of CCh and NO on ICa-L is further
supported by the following two sets of experiments. Results from the
first (n=5) are shown in Fig 9. In panels A through C,
IBMX (20 µmol/L) was used to inhibit all isoforms of PDE. In these
nystatin–perforated patch experiments, ISO and IBMX were applied
to the cell at the same time to ensure that the expected cAMP increase
would not be blunted in response to CCh. Since 1 µmol/L ISO normally
elicits a maximally effective activation of the intracellular cAMP
pathway, coapplication with IBMX did not result in a significant change
in peak ICa-L (trace b). Under
these conditions, application of CCh resulted in no appreciable
decrease in ICa-L but a significant
activation of IK(ACh) (trace c). In a second series of
experiments (n=4), milrinone (5 µmol/L), which selectively
inhibits the cGMP-inhibited PDE (PDE3), was applied at the same time as
ISO. There was no significant difference in the magnitude of
ISO-stimulated ICa-L between IBMX
and milrinone treatment. Under these conditions, subsequent application
of CCh resulted in a marked attenuation of
ICa-L (panels D through F). Thus,
these results suggest that in the specialized cardiac conduction tissue
(eg, AV node) the PDEs other than the cGMP-inhibited isoform (PDE3)
play a central role in the muscarinic cholinergic inhibition of
ICa-L.
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Discussion |
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Increased vagal tone results in various degrees of AV conduction delay or block.12 13 Our results obtained from isolated AV nodal cells from the adult rabbit heart provide evidence for the involvement of endogenous NO in this parasympathetic dromotropism. Under physiological conditions, the cholinergic attenuation of ICa-L takes place in the presence of a basal or background adrenergic tone,34 35 and this occurs at low levels of acetylcholine release that activate little, if any, outward K+ current, IK(ACh).34 35 In our experiments, adrenergic signaling was mimicked by application of ISO, and a relatively high concentration of the muscarinic agonist CCh (1 µmol/L) was used to ensure that IK(ACh) was also activated. IK(ACh), which was measured as an outward shift in the holding current (at -40 mV), provided an internal control so that in all recordings a functional muscarinic response could be verified.
For two reasons we consider that activation of IK(ACh) is
important in this signal transduction study. First, the concentration
of CCh required to activate IK(ACh) is 10 times
higher than that required to inhibit
ICa-L.35 If
IK(ACh) is activated, one can reasonably assume
that interventions (L-NMMA, methylene blue) that blunt the muscarinic
effect on ICa-L are not at the
receptor level. The presence of IK(ACh) also confirms that
our internal dialysis does not affect the agonist-receptor
interaction. Second, IBMX may block inhibitory
Gi proteins in rat adipocyte membrane.36 The
significant activation of IK(ACh) in all of our experiments
suggests that this agent and other interventions (ie, internal dialysis
alone) do not block Gi proteins in the AV nodal cells.
Anatomically, the AV node can be divided into three zones: atrial-nodal, nodal, and nodal-His.12 13 Most cells from the AV node are spindle- or rod-shaped. Whereas up to 50% of AV nodal cells may be classified as quiescent, all the cells used for the present study were spontaneously active, with mean frequency and maximum diastolic potential similar to those reported previously16 17 18 under nystatin–perforated patch recording conditions. The absence of two K+ currents, the transient outward current and the inward rectifier K+ current, from these spontaneously beating AV nodal cells minimizes any interfering currents with respect to ICa-L measurement.
Three important questions concerning the
physiological regulation of AV nodal electrical
activity (SAPs, ICa-L) have been
addressed by the present study. First, NOS3 was identified in both
single AV nodal cells and the cryostat sections of AV node. Although
recent work from other laboratories has shown that intracardiac
ganglionic cells and nerve endings innervating the AV node may contain
neuronal isoform (type I) NO synthase,23 24 the
present study demonstrates the existence of NOS3 in AV nodal and
subjacent endocardial cells using an immunohistochemical approach (Fig 1
).
The second issue was the demonstration of NO as an important mediator of muscarinic cholinergic responses in the AV node. In addition to the presence of NOS3, other evidence supporting a role of NO includes the following three observations: (1) Exogenously applied NO (SIN-1, SNAC, and SNP experiments) mimics the inhibitory effect of CCh on ISO-stimulated ICa-L. (2) Inhibition of endogenous NO production (L-NMMA experiments) blocks the CCh attenuation of ICa-L. (3) The inhibitory effect of exogenous NO on ISO-stimulated ICa-L can be abolished by prior exposure to CCh, indicating a common biochemical pathway for both NO and CCh. In fact, the muscarinic cholinergic effect on ICa-L is also blocked by L-NMMA in feline atrial myocytes.37 Furthermore, in rat myocardium both the negative inotropic effect and the stimulation of NO synthase activity and cGMP production by CCh are inhibited by L-NMMA, oxyhemoglobin, and methylene blue.38 Nevertheless, the present study does not imply that in the intact animal or organ the exclusive source of NO is from AV nodal cells. For example, NO also might be released from the nerve terminals in direct contact with AV nodal cells,23 24 under altered autonomic tone. In this case, NO could function either as a primary messenger in response to parasympathetic stimulation or as a later step in a signaling pathway after acetylcholine release.
In a variety of tissues NO is known to activate guanylyl
cyclase, which results in elevation of intracellular
cGMP.1 2 This biochemical event also takes place in
specialized cardiac myocytes, including both AV and sinoatrial nodal
cells, from rabbit heart.9 10 11 This notion is supported by
the observations that (1) the inhibitory effects of both
CCh and SIN-1 can be blocked by methylene blue (Fig 6), which is known
to interfere with NO-induced activation of guanylyl cyclase and to
decrease cGMP levels, and (2) cGMP directly inhibits ISO-stimulated
ICa-L (Fig 7). Although methylene
blue is not a perfectly specific inhibitor of guanylyl
cyclase, internal dialysis of methylene blue does not block agonist
binding to the muscarinic receptor or Gi protein
activation. This was evidenced by the activation of IK(ACh)
(Fig 6) in all of our methylene blue experiments.
Third, our findings also suggest that cGMP-dependent activation of a cAMP-specific PDE is an important step in the muscarinic cholinergic regulation of ICa-L in the mammalian AV node. A cGMP-stimulated phosphodiesterase (PDE2) that accelerates the breakdown of cAMP has been identified in the heart.32 33 Increased activity of this isoform of PDE would be expected to decrease cAMP activation of protein kinase A and, hence, to reduce the phosphorylation of an L-type Ca2+ channel subunit(s). Accordingly, the normal attenuating effect of CCh on ICa-L was blunted in the presence of (1) a nonhydrolyzable analogue of cAMP, 8-Br-cAMP, (2) a continuing high concentration of cAMP in the intracellular dialysis solution, and (3) IBMX, which inhibits all PDE isozymes, including PDE2. Other investigators also have observed that muscarinic cholinergic activation fails to suppress ICa-L either after PDE2 has been inhibited by IBMX39 or under internal dialysis with cAMP analogues.40 Our data argue against any direct inhibition of a cAMP-dependent protein kinase by CCh or modulation of biochemical events subsequent to activation of protein kinase A, eg, enhanced dephosphorylation of the L-type Ca2+ channel by phosphatases. The lack of CCh action on ISO-stimulated ICa-L in the presence of IBMX, or when ICa-L is enhanced by a high concentration (0.5 mmol/L) of cAMP or its nonhydrolyzable analogue, 8-Br-cAMP, further suggests that hydrolysis of cAMP by PDE is an important step leading to the attenuation of ICa-L under our experimental conditions, although these data do not exclude an effect of CCh on adenylate cyclase.
The muscarinic modulation of ICa-L may involve multiple biochemical pathways, depending upon the species or cardiac tissue being studied. For example, in mammalian ventricular cells from the guinea pig and rat hearts,41 42 43 44 both the cGMP-dependent protein kinase and protein phosphatases are thought to be important for the muscarinic agonist–induced reduction in ICa-L. On the other hand, in frog myocardium28 31 45 and in rabbit sinoatrial node9 10 11 and AV node, it appears that the most important mechanism is cGMP-induced stimulation of PDEs, which then selectively break down cAMP. ß-Adrenergic agonists and cAMP or its analogues are known to stimulate ICa-L by increasing both the available number of Ca2+ channels that can be activated by depolarization and the probability of channel opening with no change in single-channel conductance.31 46 47 The resulting reduction in cAMP levels attenuates ICa-L. Although the effects of muscarinic cholinergic stimulation on Ca2+ channel kinetics have not yet been thoroughly examined,31 a recent report indicates that part of the NO effects on cardiac myocytes may depend upon the redox state.48
In summary, the present study demonstrates the expression of NOS3 and an important role for NO in the cholinergic attenuation of ICa-L in single mammalian AV nodal cells. Under non–voltage-clamped conditions, NO-mediated ICa-L inhibition can lead to reduction of the frequency of SAPs and may prolong the conduction time in the specialized cardiac tissue, the AV node. The subcellular mechanism likely involves activation of the cGMP-stimulated and/or cAMP-specific PDEs.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grants HL-52320 and HL-36141 (Dr T.W. Smith).
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Footnotes |
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This manuscript was sent to Harold C. Strauss, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Received August 1, 1995; accepted February 28, 1996.
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References |
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