© 1996 American Heart Association, Inc.
Articles |
Nitric Oxide Synthase Activity in Guinea Pig Ventricular Myocytes Is Not Involved in Muscarinic Inhibition of cAMP-Regulated Ion Channels
From the Departments of Physiology and Biophysics (S.I.Z., S.P., N.R.P., R.D.H.) and Biochemistry (G.K.K.), Case Western Reserve University, Cleveland, Ohio.
Correspondence to Dr Robert Harvey, Department of Physiology and Biophysics, Case Western Reserve University, 2109 Adelbert Rd, Cleveland, OH 44106-4970. E-mail rdh3@po.cwru.edu.
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Abstract |
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Abstract It has recently been demonstrated that NO plays an obligatory role in muscarinic inhibition of ß-adrenergically stimulated ion channels in cardiac sinoatrial node cells (J Gen Physiol. 1995;106:45-65). We looked for evidence that NO might play a similar role in ventricular cells by using histochemical staining for NO synthase (NOS) activity and whole-cell patch-clamp recording of cAMP-regulated Cl- currents. Myocytes isolated from guinea pig hearts stained positively for NADPH-diaphorase activity, suggesting that these cells do express NOS. Acetylcholine (ACh) inhibition of the R(-)-isoproterenol bitartrate (Iso)–activated Cl- current was also reversed by the cGMP-lowering agents LY-83583 and methylene blue, consistent with the idea that NO activation of guanylate cyclase may contribute to muscarinic responses. However, LY-83583 and methylene blue activated the Cl- current in the presence of subthreshold concentrations of Iso alone, suggesting that their effects may not be due to antagonism of an NO/cGMP-dependent response. Furthermore, ACh inhibition of Iso-activated Cl- currents could not be mimicked by the NO donors sodium nitroprusside, 3-morpholinosydnonimine, and spermine-NO. Similarly, ACh inhibition of the Iso-activated Cl- current could not be blocked by the NOS inhibitor NG-monomethyl-L-arginine. These results indicate that even though ventricular myocytes possess NOS activity, NO production does not play an important role in muscarinic inhibition of ß-adrenergically regulated Cl- channels in these cells.
Key Words: acetylcholine • muscarinic regulation • isoproterenol • ß-adrenergic regulation • nitric oxide donors
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Introduction |
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In cardiac ventricular myocytes, activation of ß-adrenergic receptors stimulates the activity of a number of different ion currents, including those conducted by L-type Ca2+ channels and cAMP-regulated Cl- channels. The regulatory mechanism involves activation of adenylate cyclase, production of cAMP, and activation of PKA.1 In addition, the effect that ß-adrenergic stimulation has on these ionic currents can be antagonized by concurrent activation of muscarinic receptors. The mechanism of this response can be explained by muscarinic inhibition of adenylate cyclase activity through an inhibitory G protein, allowing cytosolic cAMP to return toward basal levels after breakdown by basal PDE activity.1 2 However, muscarinic stimulation also increases cGMP levels in cardiac myocytes, and it has been postulated that cGMP may be involved in the muscarinic inhibition of ß-adrenergic responses.1 This hypothesis has been supported by a number of different observations, including the fact that in frog ventricular myocytes, direct introduction of cGMP into the cytosol can antagonize cAMP-dependent activation of the Ca2+ current.3 4 This effect has been attributed to stimulation of PDE activity, since the effect can be blocked by PDE inhibitors such as IBMX. However, this type of response to cGMP is characteristic of amphibian myocytes, where activity of the predominant PDE isoform (type II) is stimulated by cGMP.5 Similar experiments in guinea pig ventricular myocytes have found that cGMP primarily has a facilitating effect on cAMP-dependent activation of Ca2+ and Cl- currents.6 7 This is presumably due to the fact that in these myocytes, the activity of the predominant isoform of PDE (type III) is inhibited by cGMP.
More recently, other cGMP-dependent mechanisms have been demonstrated to inhibit ion channel function in cardiac myocytes. This includes evidence that cGMP activation of PKG can inhibit the cAMP-activated Ca2+ current in rat ventricular myocytes.8 This mechanism has been used to explain how muscarinic agonists can inhibit the Ca2+ current activated by stimulating the cAMP/PKA pathway independent of the ß-adrenergic receptor in both frog and guinea pig ventricular myocytes.9 10 11 12 This again raises the possibility that cGMP may be involved in muscarinic inhibition of ß-adrenergic responses in mammalian myocytes. However, if this is true, an important question concerns the mechanism by which muscarinic receptor activation stimulates cGMP production. In many tissues, it has become apparent that the production of NO is a potent means of stimulating guanylate cyclase activity.13 Furthermore, it has been reported that exogenous NO can produce inhibitory effects on Ca2+ and Cl- currents in both rat and guinea pig myocytes.10 11 12 It has also been demonstrated that cardiac cells from these species possess NOS activity.14 15 16 More recently, it has been reported that NO plays an essential role in muscarinic inhibition of the ß-adrenergically stimulated Ca2+ current in rabbit cardiac pacemaker cells.17 18 The mechanism is believed to involve activation of NOS, generation of NO, activation of guanylate cyclase, production of cGMP, and stimulation of PDE activity.18 Although the same pathway has been demonstrated in frog ventricular myocytes, in frog cells, antagonism of adenylate cyclase still appears to be the primary means for muscarinic inhibition of ß-adrenergic receptor–mediated responses.1 9 19
In the present study, we examined the possibility that an NO-dependent mechanism contributes to the muscarinic regulation of ion channels in nonpacemaker mammalian cardiac myocytes. Using isolated guinea pig ventricular myocytes, we found histochemical evidence that NOS is expressed in these cells. However, ß-adrenergic and muscarinic modulation of the cAMP-regulated Cl- current was not affected by cGMP antagonists, exogenous NO, and NOS inhibitors in a manner consistent with the idea that NO plays an important role in muscarinic inhibition of ß-adrenergic responses.
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Materials and Methods |
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Cell Isolation
Ventricular myocytes were isolated using a modification of the method described previously.20 21 Briefly, hearts were excised from anesthetized adult guinea pigs and retrogradely perfused via the aorta with Krebs-Henseleit buffer containing (mmol/L) NaCl 120, KCl 4.8, CaCl2 1.5, MgSO4 2.2, NaH2PO4 1.2, NaHCO3 25, and glucose 11. The pH was maintained at 7.35 by bubbling with 95% O2/5% CO2 at 36°C. After 5 minutes, perfusion was switched to a nominally Ca2+-free solution for 5 minutes, at which time collagenase B (Boehringer Mannheim) was added to achieve a final concentration of
0.5 mg/mL. After 45 minutes of
digestion, the right ventricle was removed, cut into pieces, rinsed,
and stored in Ca2+-containing Krebs-Henseleit buffer.
Single cells were obtained by gentle trituration and used the same
day.
Voltage-Clamp Technique
Macroscopic membrane currents were recorded using the
conventional whole-cell configuration of the patch-clamp
technique.22 Micropipettes with resistances between 0.5 to
1.5 M
were used in all experiments. Electrodes were filled with an
intracellular solution containing (mmol/L) cesium glutamate 130,
tetraethylammonium chloride 15, NaCl 5,
MgATP 5, EGTA 5, Tris-GTP 0.1, and HEPES 5 (pH 7.2). The control
extracellular solution contained (mmol/L) NaCl 140, CsCl 5.4,
CaCl2 2.5, MgCl2 0.5, glucose 11, and HEPES 5.5
(pH 7.4). The bath was grounded with a 3-mol/L KCl/agar bridge;
junction potentials were not compensated for. Experiments were
conducted at 32°C, unless otherwise noted.
Cells were placed in a 0.5-mL chamber, into which control solution was introduced at a rate of 1 to 2 mL/min. However, cells were exposed to different experimental solutions using a fast-flow system as described previously.20 Using this method, the extracellular solution bathing the cell was changed in <1 s. The use of a fast-flow system allowed experiments to be completed in a short period of time, reducing the possible influence of current rundown.20
Data Acquisition and Analysis
The Cl- current was isolated by blocking all
K+ channels with Cs+-and/or TEA-containing
intracellular and extracellular solutions. L-type Ca2+
channels were blocked by adding 1 µmol/L nisoldipine to all
extracellular solutions. Any T-type Ca2+ channels and
Na+ channels were inactivated by using a
depolarized holding potential. Currents were recorded using an
Axopatch 200 voltage-clamp amplifier (Axon Instruments), filtered
at 2 to 5 kHz, and sampled at a frequency of 6.7 kHz using an
IBM-compatible computer with a TL-1-125 interface and pCLAMP software
(Axon Instruments). The time courses of changes in
Cl- conductance were monitored as drugs were washed
in and out by recording the time-independent current
elicited by 100-ms voltage steps to +50 mV once every 3 s. When the
conductance reached a steady state, current-voltage relationships
were recorded by applying 100-ms voltage steps from the holding
potential of -30 mV to test potentials from -120 to +50 mV
in 10-mV increments at a rate of one step every 200 ms. The
Cl- current was defined as the agonist-induced
difference current determined by subtracting currents recorded in
the absence of drug from currents recorded in the presence of
drug(s). Current at each potential was measured as the average current
over a 15-ms span at the end of the 100-ms step. Slope conductance was
calculated by linear regression of the current-voltage relationship
positive to the reversal potential.
Determination of NOS Activity
Evidence of NOS activity was obtained using an histochemical
approach. After isolation, myocytes were attached to microscope slides
using an adhesive (Cell-Tak, Becton-Dickinson). Attached cells were
then fixed by exposure to 4% paraformaldehyde,
followed by washing in PBS at pH 7.4. The NADPH-diaphorase
technique used was similar to that described previously.23
Fixed cells were incubated for 1 hour in PBS containing 0.3% Triton
X-100, 1 mmol/L ß-NADPH, and 0.2 mmol/L nitro blue tetrazolium. In
the presence of ß-NADPH, NOS reduces tetrazolium to formazan, which
appears as a dark (blue) stain. Controls were conducted by incubating
samples of cells isolated from the same heart in PBS containing
everything but ß-NADPH.
Drugs and Chemicals
ACh (Research Biochemicals International), histamine
hydrochloride (Sigma Chemical Co), SNP (Sigma), SIN-1 (Molecular
Probes), spermine-NO (Research Biochemicals International), MB
(Sigma), and L-NMMA (Calbiochem) were prepared as aqueous
stock solutions just before use. Iso (Research Biochemicals
International) was prepared as an aqueous stock solution containing an
equimolar concentration of ascorbic acid (Sigma). In an attempt to
prevent oxidation of Iso in NO donor–containing solutions, the
final concentration of ascorbate was increased to 100 µmol/L. All NO
donor–containing solutions were also protected from direct light.
Because the half-life of spermine-NO is only 40
minutes,24 solutions containing this compound were
prepared immediately before each experiment. LY (Research Biochemicals
International) was prepared as a stock solution in ethanol. The final
concentration of ethanol was <0.4%. In control experiments, ethanol
alone (ie, without LY) did not affect responses to either Iso or ACh.
Nisoldipine (a gift from Miles Laboratories) was prepared as a stock
solution in polyethylene glycol (Sigma). An equal concentration of
polyethylene glycol (0.05%) was present in all solutions.
Iso Concentration Measurements
Measurements of Iso concentrations in control and NO donor
(SNP)–containing solutions were obtained using a Shimadzu HPLC system
along with an electrochemical detector. The HPLC system consisted of an
Ultrasphere ODS column (4.6 mmx25 cm, 5-µm particle; Beckman), a
guard column (4.6 mmx3 cm), and a Shimadzu LC 600 pump. The mobile
phase of the HPLC system had a pH of 2.7 and contained 0.08 mol/L
NaH2PO4, 0.1 mol/L
NaNO3, 200 mg/L sodium octyl sulfate, 5 mg/L
disodium EDTA, and 4% (vol/vol) acetonitrile. The elution of Iso was
performed at 40°C with an applied potential of +0.8 V. Sample
run-through time was 40 minutes. Iso appeared as a single peak
after 33 to 35 minutes. The relative Iso concentration was
automatically determined by integration of the area under the peak.
Solutions were prepared with an initial concentration of 3 µmol/L
Iso. This is higher than the concentration used in most of the
electrophysiological experiments.
Therefore, it is possible that these measurements underestimate the
rate of Iso degradation in SNP-containing solutions.
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Results |
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If muscarinic responses in isolated ventricular myocytes do involve NO as a second messenger, then these cells should express the enzymatic activity necessary for NO production. NOS activity has been identified in isolated rat ventricular myocytes.14 16 In order to determine whether guinea pig ventricular myocytes also possess NOS activity, we used an NADPH-diaphorase assay. It has been demonstrated that NADPH diaphorase and NOS activities are different properties of the same enzyme25 and that NADPH diaphorase activity can be used as a marker for NOS.26 27 After being fixed and permeabilized, isolated guinea pig ventricular myocytes that were incubated with nitro blue tetrazolium and ß-NADPH stained positively for NOS activity (Fig 1
).
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Assuming that muscarinic agonists can stimulate NOS, NO
production would then be expected to activate
guanylate cyclase,13 leading to the
production of cGMP, and it is well documented that ACh
stimulates the production of cGMP in these cells.1
LY and MB are commonly used to block the production of cGMP by
guanylate cyclase.9 10 11 12 28 29 Therefore, we
determined whether LY and MB could antagonize ACh inhibition of the
Iso-activated Cl- current. Activation of
the Cl- current with 30 nmol/L Iso was almost
completely antagonized by concurrent exposure to 1 µmol/L ACh.
However, subsequent exposure to LY caused a concentration-dependent
reversal of the ACh effect (Fig 2). The threshold for
the response to LY was 30 µmol/L, and complete reversal of the ACh
response was observed with 300 µmol/L LY. In fact, in some
experiments, 300 µmol/L LY caused a rebound effect, where the
magnitude of the current in the presence of Iso, ACh, and LY was
greater than the magnitude of the current in the presence of Iso alone.
Experiments conducted with MB produced similar results. The response to
1 µmol/L ACh was reversed by 100 µmol/L (n=3) and 1 mmol/L (n=2)
MB.
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The fact that the effects of ACh could be blocked by both of these
inhibitors of guanylate cyclase is
consistent with the idea that the inhibitory
effects of ACh may be mediated by an NO/guanylate
cyclase-dependent pathway. However, it has been previously
demonstrated that the magnitude of the response to ACh depends on the
level of ß-adrenergic stimulation, such that increasing the level
of ß-adrenergic stimulation decreases the level of muscarinic
inhibition.20 21 Therefore, an alternative explanation for
the effects of LY and MB could be that rather than inhibiting the
response to ACh, they are actually facilitating the effect of Iso. For
this reason, we examined the effects of LY and MB on the
Cl- conductance of these cells in the absence of ACh.
In the absence of Iso and ACh, 100 µmol/L LY did not have any effect
in 12 cells, even though Cl- channels were
demonstrated to be present upon subsequent exposure to Iso.
However, in three other cells, the same concentration of LY did
activate some Cl- current. Lower
concentrations of LY (30 µmol/L; see Fig 3)
consistently (n=7) had no effect in the absence of Iso. Similar
results were obtained with MB alone. At concentrations of 30 to 100
µmol/L, MB had no effect in the absence of Iso (n=7). However, both
of these drugs significantly and consistently activated
the Cl- current in the presence of subthreshold
concentrations of Iso. Fig 3A demonstrates the facilitating effect of
30 µmol/L LY in the presence of 1 nmol/L Iso. At the end of the
experiment, each cell was exposed to a supramaximally stimulating
concentration of Iso (3 µmol/L), so that the relative magnitude of
the response to LY or MB could be determined. Fig 3B illustrates the
responses normalized to this maximal current. Neither Iso (1 nmol/L)
nor LY (30 µmol/L) alone had a significant effect, but applied
together, they activated the Cl- current with
an amplitude close to that produced by a maximally effective
concentration (3 µmol/L) of Iso alone. MB had a similar, although
somewhat weaker, facilitating effect (Fig 3C). These results suggest
that in the previous experiments (see Fig 2
) LY and MB may not have
acted to specifically antagonize the response to ACh. This then leaves
open the question of whether muscarinic inhibition of
ß-adrenergic responses is mediated by NO-dependent activation of
guanylate cyclase.
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To further test the hypothesis that muscarinic stimulation of NO
production contributes to the inhibition of ß-adrenergic
responses, perhaps by a guanylate cyclase–independent
mechanism, we examined whether it was possible to mimic the effects of
ACh with exogenous NO. For these initial experiments, we used the NO
donors SNP and SIN-1. Both compounds have been reported to inhibit the
IBMX- and Iso-activated Ca2+ current in guinea
pig ventricular myocytes.10 12 Our preliminary
results suggested that these NO donors could also effectively inhibit
the Iso-activated Cl-
current.30 In these earlier experiments, the
Cl- current was activated by exposing cells
to a solution containing 30 nmol/L Iso. The cells were then exposed to
a solution of Iso plus various concentrations of SNP or SIN-1. However,
relatively high concentrations (1 mmol/L SNP or 300 µmol/L SIN-1)
were required to observe inhibition. This suggested that the
inhibitory response might be due to a nonspecific effect.
Closer examination demonstrated that the blocking effects of these
compounds also depended on the age of the Iso and NO
donor–containing solution. We discovered that NO donors had
virtually no effect on the Iso-activated
Cl- current if the solution was used within 10 to 30
minutes after preparation. However, after 60 to 120 minutes, the effect
of the NO donors became apparent. Fig 4 illustrates
experiments on three different cells using the same set of solutions.
In the first cell (Fig 4A), SNP in an Iso-containing solution
prepared 15 minutes earlier had no discernible effect on the
Iso-activated Cl- current. When the same
solution was used 105 minutes after it had been prepared, it produced a
30% reduction in the current, which was reversible upon returning to
the solution containing Iso alone (Fig 4B). When the solution was used
150 minutes after it had been prepared, it caused complete inhibition
of the current (Fig 4C). This effect was also reversible; however, it
was reversed when the cell was exposed to an identical SNP-containing
solution that had been prepared only 7 minutes before it was used. Note
that subsequent return to the original Iso-containing solution
(without SNP) did not result in a change in the current magnitude,
suggesting that the change in response to SNP could not be explained by
a gradual decrease in the effectiveness of Iso in all solutions,
regardless of whether SNP was present. Instead, these results are
consistent with the idea that the effectiveness of Iso was
decreased only in the SNP-containing solutions. Similar results were
obtained in three cells. The same kind of response was also observed
when SIN-1 was used instead of SNP.
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Others have suggested that NO donors may cause the oxidation of
catecholamines.31 Although 100 µmol/L
ascorbic acid was included in these solutions to guard against such a
problem, we examined this question more directly by measuring the Iso
concentration in NO donor–containing solutions. Iso was measured
in solutions identical to those used in the patch-clamp
experiments. The results (Fig 5) demonstrate that the
presence of SNP did not cause a significant decrease in Iso
concentration during the first 35 minutes, but after this, there was a
time-dependent decrease. After 160 minutes, Iso was no longer
detectable. On the other hand, the concentration of Iso did not
decrease significantly over the same time period in SNP-free solutions.
These results demonstrate that NO donors such as SNP significantly
accelerate degradation of Iso, even in the presence of 100 µmol/L
ascorbic acid.
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Another approach to determine whether NO donors can antagonize the
cAMP-dependent activation of cardiac ion channels while avoiding the
problem of Iso oxidation would be to activate the current with
compounds such as histamine or forskolin, which should not be
susceptible to this kind of degradation.32 Other than
acting through an H2 histaminergic receptor, histamine
activates currents in cardiac ventricular
myocytes through the same mechanism as Iso, and these effects can be
antagonized by muscarinic receptor stimulation.33 34 Fig 6 illustrates that SNP, over a concentration range of 10
µmol/L to 1 mmol/L, had no effect on the Cl-
current activated by 300 nmol/L histamine. Even though SNP had
no effect, 1 µmol/L ACh readily inhibited the
histamine-activated current.
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ACh also inhibited the Cl- current activated
by 3 µmol/L forskolin, but 1 mmol/L SNP still had no effect (Fig 7). This result was confirmed in four separate
experiments. However, SNP is known to produce ferricyanide, in addition
to NO. Ferricyanide is an oxidizing agent, which might be expected to
produce effects of its own35 and complicate interpretation
of the results with SNP. Therefore, we also examined the response to
SIN-1, another commonly used NO donor that does not produce this
byproduct. It was found that the
forskolin-activated Cl- current was also
not inhibited by 300 µmol/L SIN-1. However, SIN-1 is known to produce
superoxide radicals (O2·), and superoxide plus NO can
combine to form peroxynitrite (OONO-). Superoxide and
peroxynitrite are also capable of producing effects of their
own,35 which could complicate interpretation of the
results with SIN-1. In an attempt to circumvent any potential problem
associated with the concurrent generation of NO and superoxide
radicals, we further examined the response to SIN-1 in the presence of
1000 U/mL superoxide dismutase (Fig 8A). However, even
under these conditions, 300 µmol/L SIN-1 still had no effect on the
forskolin-activated current. These results were confirmed
in six experiments. As a final test to determine whether exogenous NO
can mimic the inhibitory effects of ACh, we examined the
response to spermine-NO. This compound is believed to spontaneously
release NO without producing other active byproducts, and it is
approximately equipotent to SIN-1 in NO-dependent
bioassays.24 However, as with SIN-1, 300 µmol/L
spermine-NO did not significantly inhibit the Cl-
current activated by forskolin (Fig 8B). In nine separate
experiments, the Cl- conductance recorded in the
presence of forskolin plus spermine-NO was 94±7.9% of that
measured in the presence of forskolin alone.
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I) obtained by subtracting the currents recorded under
conditions illustrated in panels A and B, respectively.
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As a final test to find out if NO contributes to the muscarinic
inhibition of ß-adrenergic responses, we determined whether
L-NMMA, a competitive inhibitor of NOS, could
antagonize the response to ACh. This is the same approach used
previously to demonstrate the role of NO in muscarinic inhibition of
ß-adrenergic responses in sinoatrial node
cells.17 18 Exposure of sinoatrial node cells to 100
µmol/L extracellular L-NMMA has been shown to be
effective in as little as 10 minutes,17 and unlike some
NOS inhibitors, L-NMMA does not have to be
activated by deesterification once inside the cell. To
facilitate L-NMMA reaching an effective concentration
inside the cell, it was added to both the intracellular and
extracellular solutions. But even under these conditions, neither 200
µmol/L (n=2) nor 500 µmol/L (n=6) L-NMMA blocked the
response to ACh. To further ensure the likelihood that NOS activity was
being inhibited, the concentration of L-NMMA in the
intracellular and extracellular solutions was increased to 2 mmol/L,
and cells were preincubated in an external solution containing 2 mmol/L
L-NMMA for at least 1 hour before beginning each
patch-clamp experiment. Fig 9A illustrates that even
when this protocol was used, L-NMMA did not block the
response to ACh. To rule out the possibility that L-NMMA
might not be partially blocking the response, we compared the effect of
1 µmol/L ACh on the Cl- current activated
by 10 nmol/L Iso in the presence (n=6) and absence (n=7) of 2 mmol/L
intracellular and extracellular L-NMMA (Fig 9B). There was
no significant difference in the magnitude of the response to ACh.
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Discussion |
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NOS is classified as belonging to one of three isoforms.13 Type II was originally described in macrophages, and it is referred to as iNOS because its activity is induced by regulating gene expression. Types I and III were originally described in neuronal and endothelial cells, respectively, and each is referred to as cNOS because its activity is constitutive and not controlled by regulating gene expression. Both iNOS and cNOS have been reported in cardiac myocytes. Evidence for iNOS activity has been described in isolated rat ventricular myocytes after exposure to cytokines over a period of several hours.15 36 Although we cannot rule out the possibility that the procedure used to isolate cells in our study did not induce this type of NOS, it seems more likely that these cells express cNOS, since evidence for cNOS activity has been previously reported in cardiac myocytes. Balligand et al14 used a reporter cell bioassay to demonstrate that muscarinic stimulation of rat ventricular myocytes resulted in the production of NO. Schulz et al15 demonstrated the presence of cNOS activity in rat myocytes by measuring the production of [14C]L-citrulline, a byproduct of the formation of NO from [14C]L-arginine. It has been suggested that isolated guinea pig ventricular myocytes do not possess cNOS activity, but this was based on a bioassay method.37 More recently, Decking et al16 demonstrated cNOS activity in guinea pig myocardium using the more sensitive citrulline assay. However, their work was conducted using intact cardiac tissue, which may have included endothelial cells with endogenous cNOS activity. Our results demonstrating the presence of NADPH-diaphorase activity in isolated ventricular myocytes supports the conclusion that guinea pig ventricular myocytes do possess cNOS activity.
A characteristic feature of cNOS is that its enzymatic activity is
Ca2+ dependent. In our experiments, cells were dialyzed
with an intracellular solution containing a relatively high
concentration of EGTA, which might be expected to limit any response
involving a Ca2+-dependent mechanism. Therefore, one might
speculate that under different conditions there might be a component of
the ACh response that does involve NO production. However, this
is not consistent with the fact that exogenous NO had no effect
on the cAMP-activated Cl- current (see Figs 6 through 8). Furthermore, the EGTA-containing pipette solution used in
the present study would not necessarily be expected to block
responses involving activation of cNOS. Han and
colleagues17 18 demonstrated that muscarinic inhibition of
the ß-adrenergically stimulated Ca2+ current in
rabbit sinoatrial node cells is mediated solely by an NO-dependent
mechanism, presumably through the activation of cNOS. Although their
experiments were conducted using the perforated patch-clamp
technique, which would prevent any buffering of intracellular
Ca2+, others have demonstrated intact muscarinic
responses in the same cells dialyzed with high concentrations of
EGTA.38 39 40 This suggests that buffering the bulk of the
intracellular Ca2+ with EGTA is not able to block
NO-dependent responses. Furthermore, if buffering intracellular
Ca2+ does attenuate an NO-dependent component of the
muscarinic inhibition in guinea pig ventricular myocytes,
then ACh should have a greater effect in nondialyzed cells. However,
when responses obtained using the conventional patch-clamp
technique were compared with those obtained using the perforated
patch-clamp technique, we found that dialysis of guinea pig
ventricular myocytes with the same solution used in the
present experiments did not diminish the inhibitory
response to ACh.20
The idea that guinea pig myocytes possess NOS activity is not
particularly surprising, since it is well documented that muscarinic
stimulation results in cGMP production in cardiac muscle and
that NO is the primary activator of guanylate
cyclase activity in most mammalian cells.13 Therefore,
if muscarinic stimulation exerts an effect through the generation of
NO, it is likely to be ultimately mediated by the subsequent
stimulation of guanylate cyclase and production of
cGMP. Consistent with this idea is the observation that LY and
MB antagonized the effects of ACh. These results are consistent
with those of other groups that have demonstrated that these compounds
have a similar effect on muscarinic antagonism of the cAMP-stimulated
Ca2+ current in a variety of different cell
types.9 10 11 12 18 However, this does not necessarily mean that
LY and MB can directly antagonize the muscarinic response. Even though
LY and MB are believed to inhibit NO-dependent activation of
guanylate cyclase, both compounds were also found to
facilitate the response to ß-adrenergic receptor stimulation (see
Fig 3) and H2 histaminergic receptor stimulation (S.I.
Zakharov and R.D. Harvey, unpublished data, 1995) in the absence of
ACh. Therefore, clear interpretation of such effects is difficult. One
possible explanation is that there is a basal production of NO
and cGMP, which somehow acts to suppress the response to agonists like
Iso and histamine. LY and MB might then increase the sensitivity to
cAMP-dependent agonists by inhibiting this basal production of
cGMP. However, this is difficult to accept because cGMP has actually
been shown to have a facilitating effect on the cAMP-regulated
Cl- current in these cells.7 In this
case, inhibition of basal cGMP production would be expected to
decrease the sensitivity to Iso and histamine. Perhaps a more likely
explanation is that LY and MB exert effects other than inhibition of
guanylate cyclase. It has been suggested that they may
either directly or indirectly inhibit PDE activity,12
perhaps through the production of superoxide
ions.41 42 An effect such as this could explain the
facilitation of cAMP-dependent responses. This question requires
further investigation. In any case, the results obtained with LY and MB
cannot be used to conclusively support the idea that muscarinic
inhibitory responses involve an NO/cGMP-dependent
mechanism.
Another way to address the question of whether NO is involved in mediating muscarinic responses is to determine whether exogenous NO can mimic the effects of ACh. Mery et al9 have demonstrated that NO donors can inhibit cAMP-dependent stimulation of the Ca2+ current in frog ventricular myocytes by stimulating the production of cGMP, which then acts on a cGMP-stimulated PDE. However, as mentioned above, in guinea pig myocytes the predominant effect of cGMP is to facilitate cAMP-dependent responses.6 7 Nevertheless, SNP and SIN-1 have still been reported to inhibit the cAMP-stimulated Ca2+ current in guinea pig ventricular myocytes via a cGMP-dependent mechanism.10 12 These inhibitory effects have been attributed to the activation of a cGMP-dependent protein kinase (PKG), which has been shown to have direct inhibitory effects on cAMP-activated Ca2+ currents in rat ventricular myocytes.8 A similar mechanism has been used to explain the ability of muscarinic stimulation to inhibit currents activated by PDE inhibitors such as IBMX. Although muscarinic stimulation would still be expected to inhibit adenylate cyclase activity, in the absence of PDE activity, any current stimulated by IBMX should then be resistant to muscarinic antagonism, since there would be no way for cAMP to return to basal levels. However, muscarinic agonists like ACh and carbachol readily inhibit Ca2+ and Cl- currents stimulated by maximally effective concentrations of IBMX. It has been postulated that inhibition under such conditions is due to the ability of muscarinic agonists to stimulate the production of cGMP and activation of PKG.10 11 However, such conclusions are based in part on the ability of compounds like LY and MB to reverse the effects of carbachol.
Initial experiments with SNP and SIN-1 suggested that a similar mechanism might be involved in muscarinic regulation of the Cl- current.30 However, the fact that very high concentrations of these NO donors were required suggested that these might be nonspecific effects. Further experiments presented here support this conclusion. Any NO donor–dependent inhibition of the Cl- current that we observed was most likely due to the ability of these compounds to facilitate the degradation of Iso. This was supported by three lines of evidence. First, any effects of SNP and SIN-1 were observed only when solutions were >30 minutes old. Second, the concentration of Iso decreased in a time-dependent manner in SNP-containing solutions but not in control solutions. Third, SNP and SIN-1, even at extremely high concentrations, had no effect on the Cl- current activated by either histamine or forskolin.
The observation that SNP and SIN-1 did not inhibit the cAMP-stimulated Cl- current is interesting, because it would suggest that these compounds have different effects on Cl- and Ca2+ currents. Wahler and Dollinger12 reported that SIN-1 inhibited both the Iso- and IBMX-stimulated Ca2+ current in guinea pig myocytes. Although the effects of SIN-1 on the Iso-stimulated current might be complicated by the effects of this compound on the Iso concentration, this seems less likely to explain the inhibitory effects of SIN-1 at concentrations as low as 1 µmol/L.12 Furthermore, such an explanation does not explain the ability of SIN-1 to inhibit the IBMX-activated current. Similar results have also been reported using SNP. SNP is generally considered less effective than SIN-1 at generating NO. For this reason, significantly higher concentrations of SNP are often used. However, Levi et al10 reported that 0.1 to 10 µmol/L SNP produced significant inhibition of the IBMX-activated Ca2+ current in guinea pig ventricular myocytes. The reason that these NO donors appear to have different effects on Ca2+ and Cl- currents in cardiac myocytes is not clear. It may be related to differences in regulatory mechanisms affecting each type of channel. For example, the present results may suggest that unlike Ca2+ channels, Cl- channels are not regulated by PKG. However, another explanation may be that there are differences in the potential direct effects that NO or NO donors may have on specific channel proteins. It has recently been suggested that NO donors may have direct effects on L-type Ca2+ channels in the heart by altering the redox state of the cells.43 One conclusion that can be made, however, is that any inhibitory effect that NO may have on the Ca2+ current in these cells must not involve the cAMP-regulatory pathway that is shared by both the Cl- and Ca2+ currents.
The final test that we conducted to evaluate the potential role of NO in contributing to muscarinic inhibition of ß-adrenergically stimulated ion channels involved the use of L-NMMA. L-Arginine is the natural substrate used by NOS in the production of NO. L-NMMA is a structural analogue of L-arginine that acts as a competitive inhibitor of NOS. Han and colleagues17 18 demonstrated that L-NMMA and nitro-L-arginine methyl ester blocked muscarinic inhibition of the Iso-stimulated Ca2+ current in rabbit sinoatrial node cells without affecting the muscarinic-activated K+ conductance. A similar approach has also been used to demonstrate the role of NO in adenosine inhibition of ß-adrenergic responses in rabbit atrioventricular node cells.44 However, we were unable to observe any detectable effect of NOS inhibition on the response of the Cl- current to ACh in guinea pig ventricular myocytes. A negative response to L-NMMA applied extracellularly alone might have been explained by an inability of this compound to reach effective concentrations inside the cell, but we added L-NMMA to the intracellular as well as extracellular solutions. Furthermore, we preincubated cells with L-NMMA for >1 hour before conducting experiments, and we used concentrations of L-NMMA that were up to 20-fold greater than those shown to be effective in sinoatrial node cells.17 Therefore, our conclusion is that NOS activity is not involved in contributing to the muscarinic inhibition of cAMP-regulated Cl- channels in guinea pig ventricular myocytes. This suggests that the mechanism of muscarinic regulation of ion channels may differ depending on the type of ion channel being studied, the tissue from which the cells were isolated, and/or the animal species from which the heart was obtained.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by a grant from the National Institutes of Health (HL-45141), an Established Investigatorship from the American Heart Association (Dr Harvey), and a Fellowship from the Northeast Ohio Affiliate of the American Heart Association (Dr Zakharov). The authors thank Drs W. Kroeze and L. Hool for critically reading the manuscript.
Received August 30, 1995; accepted January 23, 1996.
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