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Vol. 294, Issue 1, 216-223, July 2000
Department of Physiology, Nippon Dental University, School of Dentistry at Tokyo, Tokyo (S.M., T.T., M.I., T.N., S.Y.); and Department of Pharmacology, Niigata University, School of Dentistry, Niigata, Japan (T.K.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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The effects of NG-monomethyl-L-arginine (L-NMMA), a nitric-oxide synthase (NOS) inhibitor, on the L-type Ca2+ current (ICa) and NO effects on NOS were determined in rat ventricular myocytes. L-NMMA (10 and 100 µM) had no significant effect on basal ICa, but in a cAMP-stimulated condition due to forskolin (1 µM) or milrinone (10 µM), a cGMP-inhibited cAMP-phosphodiesterase (PDE), L-NMMA (10 and 100 µM) concentration dependently augmented ICa. The enhancing effects of L-NMMA (10 and 100 µM) on ICa were not seen in the presence of either a nonselective inhibitor of PDE, 3-isobutyl-1-methylxanthine (20 µM), resulting in a stimulated ICa condition or a cGMP-dependent protein kinase activator, 8-bromo-cGMP (200 µM). 8-Bromo-cGMP (200 µM) inhibited 100 µM L-NMMA-induced ICa increase in the simultaneous application of forskolin (1 µM). Acetylcholine (ACh; 1 and 3 µM) inhibited 1 µM forskolin-stimulated ICa in a concentration-dependent manner, but this inhibitory action of ACh was significantly attenuated by the additional application of L-NMMA (100 µM). In the continuing presence of both L-NMMA (100 µM) and forskolin (1 µM), ACh (6 µM) had no inhibitory effect on ICa. In another series of experiments with isolated ventricular myocytes, we obtained both the positive staining of NADPH-diaphorase activity and the expression of the endothelial isoform of NOS. These data suggest that the effect of L-NMMA on ICa in a cAMP-stimulated condition with or without cholinergic inhibition is due to inhibition (acute effects) of a cGMP-stimulated cAMP-PDE via inhibition of the endothelial isoform of NOS.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The
conversion of L-arginine to L-citrulline plus
nitric oxide (NO) is produced by constitutive NO synthase (cNOS), or
after cytokine stimulation, by inducible NOS (Moncada et al., 1991
; Nathan and Xie, 1994). cNOS in saline-treated rats and inducible NOS in
rats after pretreatment with endotoxin or cytokine are found in
ventricular tissue slices as well as in isolated cardiac myocytes
(Schultz et al., 1992). Furthermore, two isoforms of cNOS have been
cloned from rat vascular endothelium (ecNOS) and brain (Nathan and Xie,
1994). The ecNOS protein expression also occurs in rat cardiac myocytes
(Balligand et al., 1995).
Administration of the NOS inhibitor
NG-monomethyl-L-arginine
(L-NMMA) causes increases in mean blood pressure
and systemic vascular resistance suppresses cardiac output in
anesthetized dogs pretreated with either saline or endotoxin (Klabunde
and Ritger, 1991). Perfusion with
L-methyl-L-arginine in an
isolated rat heart inhibits cardiac contractility in
isoproterenol-stimulated hearts and this inhibitory action is
accompanied by a decrease in both myocardial cGMP and cAMP
concentrations (Klabunde et al., 1992). In contrast,
N
-nitro-L-arginine,
one of the NOS inhibitors, potentiates positive inotropic action of
isoproterenol on electrically stimulated rat cardiac myocytes but does
not significantly alter basal contractility (Balligand et al., 1993).
In addition, Balligand et al. (1995) reported that
L-NMMA attenuated the inhibitory effects of
carbamylcholine on the increase in contractility induced by
isoproterenol. Nawrath et al. (1995), however, demonstrated that
L-NMMA failed to influence the muscarinic effect
on the force of contraction or frequency in rat and guinea pig hearts.
Therefore, contradictory opinions remain as to how NOS inhibitors
influence myocardial contractility in the 
-adrenergically stimulated
condition with or without the activation of muscarinic receptors.
cGMP is known to regulate both cGMP-dependent protein kinase (PKG;
Lincoln and Corbin, 1983) and phosphodiesterase (PDE; Whalin et al.,
1988). The physiological action of NO donors generally occurs as a
result of the activation of soluble guanylyl cyclase (Katsuki et al.,
1977). In mammalian myocardium, a PKG plays a major role in the
cGMP-induced decrease in calcium current (ICa; Levi et al., 1989;
Méry et al., 1991, 1993). Indeed, the inhibitory effect of an NO
donor molsidomine is thought to be related to the activation of PKG in
rat ventricular myocytes (Matsumoto, 1997). In frog ventricular
myocytes, 3-morpholine-syndnonimine (SIN-1), one of the metabolic
products of molsidomine, inhibits ICa by accumulating intracellular
cGMP to activate a cGMP-stimulated cAMP-PDE (Méry et al., 1993).
The resulting reduction in the cAMP level due to the activation of a
cGMP-stimulated cAMP-PDE is responsible for NO-mediated cholinergic
inhibition of ICa in isolated primary pacemaker cells from the rabbit
sinoatrial node (Han et al., 1995). The question arises whether the
effect of NOS inhibitors on ICa is the result of inhibition of PKG or
cGMP-stimulated cAMP-PDE.
To elucidate the mechanism of the NOS inhibitor effect on rat cardiac
myocytes, L-NMMA that does not inhibit muscarinic receptors (Buxton et al., 1993) and that is not necessary for de-esterification (Han et al., 1994) was used. We therefore investigated the effects of
L-NMMA on the L-type ICa responses to the PKG activator
8-bromo-cGMP (8-Br-cGMP; Geiger et al., 1992; Lincoln and Cornwell,
1993) and a cAMP-stimulated condition due to forskolin,
3-isobutyl-1-methylxanthine (IBMX, a potent inhibitor of
cGMP-stimulated PDE; Levi et al., 1989), or milrinone (a specific
inhibitor of the cGMP-inhibited PDE; Nicholson et al., 1991) as well as
the acetylcholine (ACh)-induced ICa inhibition in the presence of
forskolin with or without L-NMMA application. We also
examined the effects of 8-Br-cGMP on ICa responses to simultaneous
administration of L-NMMA and forskolin. In another series
of experiments with NADPH-diaphorase assay and Western blotting
analysis, we examined whether the induction of a protein corresponding
to ecNOS is occurring in isolated rat ventricular myocytes.
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Materials and Methods |
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Introduction
Materials and Methods
Results
Discussion
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Animal Preparations and Electrophysiological Experiments.
Single cardiac cells were isolated from the ventricles of rat hearts as
described in Matsumoto (1997). Briefly, rats weighing 200 to 300 g
were anesthetized by inhalation of ether vapor in a chamber or with
i.p. administration of pentobarbital sodium. Then the heart was rapidly
removed and perfused via the aorta with Langendorf apparatus. To obtain
single ventricular cells the heart was perfused with
Ca2+-free Tyrode's solution containing
collagenase (5-10 mg/50 ml; Wako Pure Chemical, Tokoyo, Japan) for 20 min.
. Current was measured with an amplifier (Nihon
Koden CEZ-2300). A seal was formed with an internal solution in a
pipette (1.2-2.3 M
). Then a higher negative pressure was applied
inside the pipette to rupture the patch membrane to establish the
whole-cell mode. The current-voltage (I-V) relationship was first
monitored by using step pulses (200 ms) from the holding potential
(
60 mV) to +60 mV at a frequency of 0.1 Hz. The holding potential
level was adjusted to 40 mV to avoid contamination of the
Na+-channel current. In most experiments, the
cells were depolarized every 10 s from a 40-mV holding potential
to 0 mV for 200 ms, and K+ currents were blocked
by replacing all K+ with intracellular and
extracellular Cs+. The data were acquired on-line
with an NEC PC-980/RX computer. Experiments were performed at
33-37°C.
Solutions and Drugs. The normal Tyrode's solution contained 140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 0.33 mM NaH2PO4, 5.5 mM glucose, 5 mM HEPES (Wako Pure Chemicals), and 4 mM CaCl2. The pH was adjusted to 7.4 with NaOH. The KB solution consisted of 70 mM KOH, 50 mM glutamic acid (Wako Pure Chemicals), 40 mM KCl, 3 mM MgCl2, 20 mM taurine (Wako Pure Chemicals), 20 mM KH2PO4, 10 mM glucose, 5 mM EGTA (Wako Pure Chemicals), and 10 mM HEPES. The pH was adjusted to 7.4 with KOH. The internal solution contained 20 mM NaCl, 90 mM CsOH (Sigma Chemical Co., St. Louis, MO), 40 mM aspartic acid (Wako Pure Chemicals), 5 mM ATP magnesium salt (Sigma Chemical Co.), 3 mM MgCl2, 5 mM potassium creatine phosphate (Funakoshi, Tokyo, Japan), 20 mM HEPES, 0.3 mM Na2GTP (Sigma Chemical Co.), and 20 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (Wako Pure Chemicals). The pH was adjusted to 7.2 with CsOH. CaCl2 (6 mM) was added.
L-NMMA, 8-Br-cGMP, forskolin, IBMX, and milrinone were purchased from Sigma Chemical Co. ACh was obtained from Daiichiseiyaku (Tokyo, Japan). These drugs were dissolved in either normal Tyrode's solution or ethanol as a 10 mM stock solution. All the stock solutions were kept in glass containers.Data Analysis. Before external application of L-NMMA the amplitude of basal ICa was measured, averaged, and normalized as the control. The maximal changes in the amplitude of whole-cell ICa were measured after external application of L-NMMA (10 and 100 µM) and expressed as percentage of changes in the control. The statistical difference in the concentration-dependent effects of L-NMMA on the changes in basal ICa was calculated by a one-way ANOVA for repeated measurements. The maximal changes in ICa were measured after external application of L-NMMA (10 and 100 µM) in the absence or presence of 8-Br-cGMP (200 µM), forskolin (1 µM), IBMX (10 µM), and milrinone (10 µM). Similarly, maximal changes in ICa seen after external application of ACh (1 and 3 µM) were compared in the presence of forskolin (1 µM) and forskolin (1 µM) plus L-NMMA (100 µM). Furthermore, maximal changes in ICa induced by simultaneous application of both L-NMMA (100 µM) and forskolin (1 µM) were compared in the absence or presence of 8-Br-cGMP (200 µM) or ACh (6 µM). The maximal changes in basal ICa induced by L-NMMA before or during the application of 8-Br-cGMP, forskolin, IBMX, or milrinone; the ACh-induced maximal decreases in a forskolin-stimulated ICa before and after L-NMMA application; and the L-NMMA-induced maximal increases in a forskolin-stimulated ICa before and after the application of 8-Br-cGMP or ACh were analyzed by a paired t test. All values were expressed as the mean ± S.E. A value of P < .05 was statistically significant.
NADPH-Diaphorase Assay.
The NADPH-diaphorase assay
technique, as described in Prabhakar et al. (1993), was used to
determine whether rat ventricular myocytes express NOS activity. After
isolation, myocytes were collected into 0.5-ml Eppendorf tubes that
contained Tyrode's solution in the absence or presence of 100 µM
L-NMMA, and they were incubated at 36°C for 10 to 30 min.
After suction of the solution, the cells were then fixed with 4%
paraformaldehyde and washed in PBS (pH 7.4). Fixed cells were incubated
for 2 h in PBS containing 0.3% Triton X-100 and 0.2 mM nitroblue
tetrazolium, either in the absence (control) or presence of 1 mM
-NADPH. In the presence of -NADPH, NOS reduced tetrazolium to
formazan, which appeared as a dark blue stain.
Western Blotting.
Cardiac cells were studied in Tyrode's
solution. The cells were prepared and treated for 10 to 30 min. At the
end of the treatments, the medium was aspirated, and cells were lysed
in ice-cold Laemmli buffer [50 mM Tris-HCl, 2% dithiothreitol, 2%
SDS, 0.1% bromophenol blue, and 10% glycerol (pH 6.8)] as described
in Bradford (1976). Before electrophoresis, samples were boiled for 5 min at 100°C and then centrifuged for 10 min at 10,000g at
4°C to remove insoluble materials. Protein concentrations at each
group were determined by Bio-Rad protein assay (Laemmli, 1970) and
varied <10%. Aliquots of samples (50 µg of protein) were applied to
SDS-polyacrylamide gel electrophoresis (8% linear-gel; Laemmli, 1970),
and separated proteins were transferred onto a nitrocellulose membrane
(0.4-µm pore size; Amersham, Buckinghamshire, UK) by electroblotting
on ice at 100 V for 70 min (Towbin et al., 1979). The uniformity and
completeness of protein transfer were established by staining the
membrane with Ponseau S (Sigma Chemical Co.). Immunoblotting was
performed by incubating blots for 1 h at room temperature with
blocking buffer [10 mM Tris-HCl, 150 mM NaCl, 1% Tween 20, and 3%
BSA (pH 7.6, fraction v; Sigma Chemical Co.)] and subsequently probing
with a monoclonal antibody to anti-ecNOS (1:6000; Transduction Laboratories, Lexington, KY) in blocking buffer overnight or for 1 h at 4°C. Antibody bound to ecNOS was detected with horseradish peroxidase-protein A (1:8000; Zymad Laboratory, South San Francisco, CA) in blocking buffer and visualized by the Amersham enhanced chemiluminescence system.
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Introduction
Materials and Methods
Results
Discussion
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Effects of L-NMMA on NADPH-Diaphorase Activity and
ecNOS Protein Expression of Isolated Ventricular Myocytes.
As
shown in Fig. 1, the NADPH-diaphorase
assay technique demonstrated the presence of NOS activity in isolated
rat ventricular myocytes. The cells prepared in the absence of
-NADPH (Fig. 1A) failed to exhibit a positive staining reaction, and
the cells treated in the presence of -NADPH stained positively (Fig.
1B). Similar results were obtained in 10 cells isolated from 3 hearts. However, cardiac myocytes constitutively expressed in ecNOS contained a
130-kDa protein (Fig. 1C). Similar results were obtained in ventricular
myocytes from four hearts.
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Effect of L-NMMA on Basal ICa.
Typical examples of
L-NMMA (100 µM) effects on the ICa seen in step pulses
are shown in Fig. 2A. Activation of ICa
usually started at 40 mV and reached a maximum at 0 mV. Figure 2B
presents the changes in I-V relationships of ICa from five cardiac
myocytes in the control solution and after L-NMMA (10 and
100 µM) application. L-NMMA did not shift either to the
right or the left on the step pulse-induced I-V curves. Figure 2B shows
the changes in the basal ICa seen in step pulses in response to
external application of L-NMMA at two concentrations (10 and 100 µM). L-NMMA had a weak stimulatory effect on the
basal ICa. The changes in ICa induced by L-NMMA at 10 and
100 µM are summarized in Fig. 2C. The mean value for basal ICa was
672 ± 49 pA (n = 8). L-NMMA
(10 and 100 µM) had no significant effect on basal ICa.
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Effects of L-NMMA on ICa before and after
8-Br-cGMP.
External application of 8-Br-cGMP (200 µM) that
strongly and selectively activates PKG (Geiger et al., 1992; Lincoln
and Cornwell, 1993) inhibited basal ICa but did not significantly alter
the change in basal ICa in response to L-NMMA (100 µM;
Fig. 3A). The mean (n = 5) peak ICa amplitude in the presence of 8-Br-cGMP was approximately
91% relative to basal ICa. L-NMMA (10 and 100 µM), when added after 8-Br-cGMP, had no significant effect on ICa
(Fig. 3B).
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Effect of 8-Br-cGMP on ICa Response to Simultaneous Application of
Both L-NMMA and Forskolin.
Simultaneous application of
both L-NMMA (100 µM) and forskolin (1 µM) stimulated
basal ICa by approximately 75%. In the continuing presence of both
L-NMMA and forskolin, 8-Br-cGMP (200 µM) inhibited L-NMMA-induced ICa stimulation (Fig.
4A). The applications of L-NMMA (100 µM) and forskolin (1 µM) increased basal
ICa, and under these conditions, 8-Br-cGMP significantly attenuated the increase in ICa induced by L-NMMA application in a
cAMP-stimulated condition due to forskolin (Fig. 4B).
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Effects of L-NMMA on ICa before and after
Forskolin.
Figure 5A shows the
changes in basal ICa in response to external application of
L-NMMA (100 µM) before and after forskolin application (1 µM). L-NMMA had a weak stimulatory effect on the basal
ICa. In the presence of forskolin (1 µM) that potentiated the
amplitude of basal ICa by approximately 53%, additional application of
L-NMMA (100 µM) noticeably potentiated
forskolin-stimulated ICa. Figure 5B summarizes the effects of two
concentrations of L-NMMA (10 and 100 µM) before and after
external application of forskolin (1 µM). L-NMMA
concentration dependently augmented forskolin-stimulated ICa.
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Effects of L-NMMA on ICa before and after IBMX.
IBMX (10 µM), a nonselective PDE inhibitor, potentiated basal ICa by
approximately 59%. In the continuing presence of IBMX, L-NMMA (100 µM) did not significantly alter IBMX-induced
ICa stimulation (Fig. 6A). Although
L-NMMA (10 and 100 µM) had a weak stimulatory effect on
basal ICa before IBMX application, such an effect was not observed in
the presence of IBMX (Fig. 6B).
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Effects of L-NMMA on ICa before and after
Milrinone.
Milrinone (10 µM) potentiated basal ICa by
approximately 71%, and a weak stimulatory action of L-NMMA
(100 µM) in the absence of milrinone was significantly augmented by
external application of milrinone (Fig.
7A). As shown in Fig. 7B, the stimulatory
effect of L-NMMA on ICa occurred in a cAMP-stimulated
condition closely related to the inhibition of cGMP-inhibited cAMP-PDE.
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Effect of L-NMMA on ACh-Induced ICa Inhibition in
Forskolin-Stimulated ICa.
When cardiomyocytes were exposed to 1 µM forskolin, forskolin increased basal ICa by approximately 64%,
and under these conditions, additional application of ACh (3 µM)
greatly reduced forskolin-stimulated ICa. External application of
L-NMMA (100 µM) that further increased the ICa stimulated
by 1 µM forskolin significantly attenuated the inhibitory action of
ACh (1 µM; Fig. 8A). As shown in Fig. 8B, the changes in ACh (1 and 3 µM)-induced ICa decreases in the forskolin (1 µM)-stimulated condition before and after external application of L-NMMA at two concentrations (10 and 100 µM) were compared. L-NMMA attenuated the decrease in ICa
induced by ACh application in a cAMP-stimulated condition due to
forskolin.
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Effect of ACh on ICa Response to Simultaneous Application of Both
L-NMMA and Forskolin.
Simultaneous application of both
L-NMMA (100 µM) and forskolin (1 µM) potentiated basal
ICa by approximately 73%, and additional application of ACh (6 µM)
no longer attenuated ICa (Fig. 9A). No
inhibitory action of ACh (6 µM) on ICa was observed in the presence
of both L-NMMA and forskolin (Fig. 9B).
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Discussion |
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Abstract
Introduction
Materials and Methods
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Discussion
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This study provided evidence that L-NMMA, an NOS inhibitor, attenuated the ACh-induced ICa decrease in a cAMP-stimulated condition due to forskolin, and that L-NMMA did not potentiate ICa when IBMX, a nonselective inhibitor in several PDEs, raised the cAMP level. Because the presence of NADPH-diaphorase assay and the expression of ecNOS protein (130 kDa) were identified in isolated rat cardiac myocytes, it is possible that inhibition of NOS blocks the cholinergic attenuation of ICa in a cAMP-stimulated condition and that this blocking action is mediated by inhibition of cGMP-stimulated cAMP-PDE, which can hydrolyze cAMP. This hypothesis was further confirmed by evidence demonstrating that both forskolin and milrinone potentiated the effect of L-NMMA on ICa, whereas the ICa response to L-NMMA was not significantly altered by application of 8-Br-cGMP.
NADPH-diaphorase and NOS activities are thought to reflect different
properties of the same enzyme (Hope et al., 1991), but NADPH-diaphorase
assay has been used as a marker for NOS (Klimaschewski et al., 1992).
In this study, the presence of NADPH-diaphorase activity was found in
isolated rat cardiac myocytes. The expression of ecNOS protein (130 kDa) also was identified. The results are in agreement with a report
showing that single myocytes obtained from rat ventricles can express
the endothelial isoform of NOS (Balligand et al., 1995).
Because the NOS inhibitor L-NMMA does not block muscarinic
receptors commonly expressed in mammalian cardiovascular tissues (Buxton et al., 1993), one can expect that the appearance of
L-NMMA effects on both the basal ICa and the ACh-induced
ICa inhibition in a cAMP-stimulated condition is mediated by the
blocking action of NOS. Because NOS influences the NO-cGMP pathway, the
effect of L-NMMA on basal ICa would show opposite reactions
compared with that observed after application of NO donors. In isolated frog ventricular myocytes, the NO donor SIN-1 has no effect on basal
ICa (Méry et al., 1993). In addition, Balligand et al. (1993)
reported that carbachol, one of the muscarinic receptor agonists,
elicited NO generation from cardiac myocytes, whereas molsidomine and
nitroprusside failed to elicit NO generation. Matsumoto (1997),
however, demonstrated that molsidomine, a precursor of SIN-1,
concentration dependently inhibited the basal ICa amplitude. Furthermore, two NO-generating agents, isosorbide dinitrate and nitroprusside, in the absence of a cAMP-stimulated condition can reduce
basal ICa in single myocytes from guinea pig ventricles (Yoshinaga,
1994). This difference may imply that products of endogenous NOS are
different from NO that is liberated by exogenous vasodilator drugs
(Myers et al., 1990). Gallo et al. (1998) reported that the two NOS
inhibitors, L-NMMA (1 mM) and
NG-nitro-L-arginine
(1 mM), caused a rapid increase in the ICa obtained from guinea pig
ventricular myocytes. Presumably, conflicting data also are involved in
the species differences. In this study, L-NMMA
(10 and 100 µM) tended to increase ICa but this increase was not
significant compared with the control ICa amplitude. When external
application of forskolin increased intracellular cAMP levels,
L-NMMA application (10 and 100 µM) resulted in
a significant increase. This finding suggests that the stimulating
effect of L-NMMA on basal ICa appears only when
the level of cAMP is already high. Indeed, we found that
L-NMMA (10 and 100 µM) caused a further stimulation of milrinone-enhanced ICa. Milrinone that is a specific inhibitor of the cGMP-inhibited cAMP-PDE (Nicholson et al., 1991) is
expected to increase cAMP levels. These findings are basically consistent with a report demonstrating that inhibition of endogenous NO
with NOS inhibitors potentiates the amplitude of shortening, in
response to -receptor agonists, of freshly isolated ventricular myocytes from normal rats (Balligand et al., 1993).
In the study to measure the rat cardiac ICa, external application of
L-NMMA (1 mM) or internal dialysis with this NOS inhibitor at the same concentration attenuated the ACh-induced inhibition in the
isoproterenol-stimulated ICa (Balligand et al., 1995). With guinea pig
ventricular myocytes, Stein et al. (1993), however, reported that the
increase in cGMP content and the contractile response to carbachol were
not mediated by endogenous NO formation from L-arginine. In
the same species, Zakharvo et al. (1996) found that NOS inhibition did
not result in any detectable change in the response of cAMP-regulated
Cl
current to ACh in a -adrenergically
stimulated Cl current. From these observations,
the different types of ion channels and species differences may
contribute to the conflicting data. Furthermore, there are conflicting
data on the muscarinic regulation of -adrenergically stimulated ICa
in ecNOS knockout mice (Han et al., 1998; Vandecasteele et al., 1999).
In this study, additional application of L-NMMA (100 µM)
to the forskolin-stimulated ICa could attenuate ACh-induced inhibition,
and simultaneous application of both forskolin (1 µM) and
L-NMMA (100 µM) abolished the ICa response to ACh (6 µM). The cholinergic inhibition would occur as a result of the
L-NMMA-induced ICa increase in a cAMP-stimulated condition.
The cholinergic regulation of ICa in a cAMP-stimulated condition is
thought to be mediated by two different biochemical pathways. In one
pathway, PKG can reduce ICa in mammalian cardiac cells (Levi et al.,
1989; Méry et al., 1991) and in the other pathway, activation of
a cGMP-stimulated cAMP-PDE selectively breaks down cAMP (Méry et
al., 1993). We found that the nonselective PDE inhibitor IBMX
preventing the resulting reduction in cAMP levels had an inhibitory
effect on L-NMMA-induced ICa change. This finding suggested
that the cholinergic inhibition of ICa in a cAMP-stimulated condition
is due to cGMP-stimulated cAMP-PDE, which can hydrolyze cAMP and
inhibit cAMP-dependent phosphorylation of ICa channels. This suggestion
was further confirmed because 8-Br-cGMP did not significantly alter the
changes of ICa in response to L-NMMA application (10 and
100 µM). We also found that external application of 8-Br-cGMP could
inhibit the L-NMMA-induced ICa increase in the simultaneous
application of forskolin. Because 8-Br-cGMP selectively activates PKG
and is insensitive to breakdown by PDE (Geiger et al., 1992; Lincoln
and Cornwell, 1993), this finding probably implies that the inhibitory
effect of 8-Br-cGMP on ICa in the presence of both L-NMMA
and forskolin is independent of the cGMP-stimulated cAMP-PDE pathway.
Accordingly, the data from this study lead us to suggest that the
modification of both cholinergic and -adrenergic influences caused
by L-NMMA would not be mediated by the biochemical events
subsequent to inhibition of PKG.
In the myocardium, a pertussis toxin-sensitive G protein, Gi, that is
coupled to M2 receptors may mediate the indirect
action of ACh (Hescheler et al., 1986; Fischmeister and Shrier, 1989; Nakajima et al., 1990). Although high concentrations of the 
i subunit of Gi directly inhibit adenylyl cyclase, this inhibitory action
is only partial and depends on the subtype of this enzyme (Taussig et
al., 1993). The 
-subunits from Gi directly inhibit the
type I brain adenylyl cyclase, but these isoforms have not been
identified in the heart (Tang and Gilman, 1991). The exact mechanism by
which Gi inhibits cardiac calcium current remains to be determined.
In conclusion, our results demonstrate that the application of L-NMMA in a cAMP-stimulated condition could modify both basal ICa and ACh-induced ICa inhibition and that the induction of ecNOS protein expression occurs in isolated rat cardiac myocytes. These results suggest that ecNOS in rat cardiac myocytes may mediate NO production.
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Footnotes |
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Accepted for publication April 5, 2000.
Received for publication December 21, 1999.
Send reprint requests to: Dr. Shigeji Matsumoto, Department of Physiology, Nippon Dental University, School of Dentistry at Tokyo, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan.
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Abbreviations |
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NO, nitric oxide; cNOS, constitutive NO synthase; iNOS, inducible NOS; ecNOS, constitutive endothelial NOS; L-NMMA, NG-monomethyl-L-arginine; PKG, cGMP-dependent protein kinase; PDE, phosphodiesterase; ICa, calcium current; SIN-1, 3-morpholine-syndnonimine; 8-Br-cGMP, 8-bromo-cGMP; IBMX, 3-isobutyl-l-methyl-xanthine; ACh, acetylcholine; I-V, current-voltage.
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Abstract
Introduction
Materials and Methods
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Discussion
References
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oubunits.
Science (Wash DC)
254:
1500-1503-adrenergic regulation of heart rate, force of contraction and calcium current is preserved in mice lacking endothelial nitric oxide synthase.
Nat Med
5:
331-334[Medline].This article has been cited by other articles:
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  H. Wang, M. J. Kohr, D. G. Wheeler, and M. T. Ziolo Endothelial nitric oxide synthase decreases {beta}-adrenergic responsiveness via inhibition of the L-type Ca2+ current Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1473 - H1480. [Abstract] [Full Text] [PDF]
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 M. T. Ziolo and D. M. Bers The Real Estate of NOS Signaling: Location, Location, Location Circ. Res., June 27, 2003; 92(12): 1279 - 1281. [Full Text] [PDF]
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) and in the presence of L-NMMA at 10 µM
(
) and 100 µM (
). Vertical bars show the mean ± S.E.
(n = 5). B, effects of L-NMMA on the
basal ICa obtained by step pulses from 
) and after (
) 8-Br-cGMP (200 µM). Vertical columns show the mean ± S.E.
(n = 6).
