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Circulation Research. 1996;78:998-1008

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(Circulation Research. 1996;78:998-1008.)
© 1996 American Heart Association, Inc.

Articles

Nitric Oxide Synthase (NOS3)–Mediated Cholinergic Modulation of Ca2+ Current in Adult Rabbit Atrioventricular Nodal Cells

X. Han, L. Kobzik, J.-L. Balligand, R.A. Kelly, T.W. Smith

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.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
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 ({approx}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{Omega}. 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 {approx}-10 mV was corrected electronically. The electrode resistance was between 2 and 4 M{Omega}. 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.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
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 1Down, 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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  		Figure 1. Immunohistochemical detection of NOS3 in isolated AV nodal cells and cryostat sections of AV node. Immunostaining of isolated AV nodal cells with monoclonal anti-NOS3 shows positive labeling of cells with characteristic morphology (a and b). No specific labeling is seen with nonspecific mouse IgG (c). AV nodal cells in situ are also labeled by anti-NOS3 (d), whereas no specific labeling is seen with control IgG (e). Cells in the cryostat tissue section can be divided into three layers: the endothelial surface in the top left corner (highlighted by arrow in panel d), the central nodal cells (C) in the lower right corner (arrow), and the transitional cells (T) in between. Immunoperoxidase labeling of paraformaldehyde-fixed adherent cells or cryostat tissue sections was performed using a peroxidase-antiperoxidase detection technique (see "Materials and Methods"). Bars=20 µm (a, b, d, and e) and 40 µm (c).

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 2Down 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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  		Figure 2. Effects of NO donor SIN-1 (0.1 mmol/L) on the frequency of SAPs of single AV nodal cells. Traces of SAPs from panels A through E were recorded consecutively under the control condition (A), ISO (1 µmol/L) stimulation (B), SIN-1 in the presence of ISO (C), washout of SIN-1 (D), and washout of ISO (E), with the nystatin-perforated patch under current clamp. Horizontal lines indicate the 0-mV level. Calibration in panel C applies to all panels. Exposure to SIN-1 resulted in a reversible decrease in the frequency of SAPs (compare panels C and D with panel B) and a small but significant reduction of the action potential amplitude after ISO stimulation (n=6; see text for details).

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 3Down, 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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  		Figure 3. Effects of the NO donor SIN-1 on ICa-L. The values of ICa-L in this and all following figures were measured as the difference between the peak inward current and the holding current. In this and all following figures, the time courses of changes of ICa-L ({bullet}) and holding current ({circ}) 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 4Up, the nystatin–perforated patch technique was used. Note that 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 ISO and SIN-1. Traces labeled a, b, and c were recorded under the control condition, ISO stimulation, and SIN-1 application in the presence of ISO, respectively. Results shown in this figure are representative of observations made in five cells. *Significantly different from the ISO-stimulated group.

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 4Down 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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  		Figure 4. Effects of CCh on the ISO-stimulated ICa-L. A, Time course of changes in ICa-L ({bullet}) and holding current ({circ}) in response to test agents. B, Original currents recorded at the time points marked in panel A. C, Bar plot showing averaged changes in peak ICa-L in response to the sequential application of ISO, CCh, and SIN-1 (n=3). ICa-L was first augmented by application of the ß-adrenergic agonist ISO (1 µmol/L). Subsequent application of CCh resulted in a marked inhibition of ICa-L, which was not further reduced by SIN-1. The outward shift in the holding current reflects the activation of IK(ACh) (see text). Current traces labeled a through d were recorded in control, ISO, ISO+CCh, and ISO+CCh+SIN-1 solutions, respectively. *Significantly different from the ISO-stimulated group.

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 5Down. 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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  		Figure 5. Internal dialysis with a NO synthase inhibitor (L-NMMA, 0.5 mmol/L) blocks the inhibitory effect of CCh on ICa-L. Panels A through C show the results obtained when L-NMMA was not included in the pipette. Panels D through F illustrate the results obtained when L-NMMA was dialyzed into the cells. A and D, Time course of changes in ICa-L ({bullet}) and holding current ({circ}) in response to ISO and CCh. B and E, Current traces recorded at the time points denoted in panel A. C and F, Bar plots showing quantified changes in peak ICa-L as the percentage of control. When NO synthase was inhibited, CCh (1 µmol/L) no longer suppressed the ISO-stimulated ICa-L but still activated a significant IK(ACh). Current traces labeled a through c were recorded under control conditions, ISO stimulation, and CCh+ISO stimulation, respectively, in the presence of continuing internal dialysis. *Significantly different from the ISO-stimulated group.

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 6Down. 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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  		Figure 6. Methylene blue (20 µmol/L) blocks both SIN-1– and CCh (1 µmol/L)–induced inhibition of ICa-L. A through C, Methylene blue blocks the action of the direct NO donor SIN-1 on ICa-L (n=4). These experiments were performed using perforated-patch recordings. D through F, Methylene blue blocks the CCh-induced inhibition of ISO-stimulated ICa-L (n=5). In these experiments, methylene blue was dialyzed into the cell using the ruptured-membrane method. Superimposed current traces in panels B and E were recorded under control conditions (a), ISO stimulation (b), and SIN-1 or CCh application in the presence of ISO (c), respectively. The bar plots in panels C and F show the averaged changes in peak ICa-L in response to each test agent(s). Note that a significant activation of IK(ACh) (outward shift in the holding current) in the presence of methylene blue indicates that methylene blue does not interact directly with the M2 receptor or Gi protein. Atropine (ATR) quickly reverses the CCh activation of IK(ACh) without significantly affecting the peak ICa-L.

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 7Down. 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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  		Figure 7. Effect of internal dialysis with cGMP on ISO-stimulated ICa-L. Cells were exposed to 1 µmol/L ISO for 5 minutes before rupturing the membrane. A, Beat-by-beat decrease in ICa-L was seen immediately after starting dialysis, and the amplitude of ICa-L reached a steady state in 6 minutes. Holding current remained unchanged throughout the experiment. B, Current traces recorded at the time points marked in panel A are as follows: a, the first current trace obtained after rupturing the cell membrane; b, current trace recorded after the decrease in ICa-L reached plateau. C, Bar graph shows the quantified ICa-L inhibition by cGMP (10 µmol/L). *Significantly different from the ISO-stimulated group.

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 8ADown. 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 8BDown). 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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  		Figure 8. Effects of CCh on ICa-L stimulated by high concentrations (0.5 mmol/L) of 8-Br-cAMP (A, n=5) or cAMP (B, n=4). Superimposed current traces were recorded immediately after the onset of internal dialysis with 8-Br-cAMP or cAMP (a), 3 minutes after dialysis (b), and after CCh application in the continuing dialysis of 8-Br-cAMP or cAMP (c). Significant augmentation in peak ICa-L was seen with dialysis of both compounds, and the beat-by-beat increase normally reached a plateau 3 minutes after the onset of internal dialysis. Subsequent addition of CCh (1 µmol/L) did not attenuate ICa-L, although it did activate IK(ACh).

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 9Down. 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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  		Figure 9. Effects of two PDE inhibitors, IBMX (20 µmol/L, n=5; A through C) and milrinone (MRN, 5 µmol/L, n=4; D through F) on CCh actions. Panels A and D show the time course of changes in peak ICa-L ({bullet}) and holding current ({circ}) in response to individual test agents. Panels B and E illustrate the current traces recorded at the time points denoted in panels A and D, respectively. These superimposed current records were obtained under control conditions (a), exposure to either IBMX or MRN in the presence of ISO (b), and subsequent application of CCh in the presence of both ISO and the PDE inhibitors (c). Panels C and F represent the averaged changes in peak ICa-L in response to different test agents. In these experiments, a saturating concentration of ISO (1 µmol/L) was added with the PDE inhibitors, giving an increase in ICa-L similar to that seen with ISO alone. CCh (1 µmol/L) did not attenuate ICa-L in the presence of IBMX (A), but it still caused a significant reduction of ICa-L in the presence of MRN (B). Addition of IBMX or MRN did not affect CCh activation of IK(ACh), suggesting that these agents do not block the muscarinic receptors or inhibit the receptor-coupled Gi protein. *Significantly different from the ISO-stimulated group.


*    Discussion
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
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 {approx}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 1Up).

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 6Up), 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 7Up). 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 6Up) 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.


*    Selected Abbreviations and Acronyms
 
AV = atrioventricular
CCh = carbamylcholine
IBMX = 3-isobutyl-1-methylxanthine
ICa-L = L-type Ca2+ current
IK(ACh) = acetylcholine-sensitive K+ current
ISO = isoproterenol
L-NAME = nitro-L-arginine methyl ester
L-NMMA = NG-monomethyl-L-arginine monoacetate
NOS3 = endothelial constitutive NO synthase
PDE = phosphodiesterase
SAP = spontaneous action potential
SIN-1 = 3-morpholinosydnonimine
SNAC = S-nitroso-acetylcysteine


*    Acknowledgments
 
This study was supported by National Institutes of Health grants HL-52320 and HL-36141 (Dr T.W. Smith).


*    Footnotes
 
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.


*    References
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 up arrowIntroduction
 up arrowMaterials and Methods
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 *References
 

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