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MS 11464 Received 24 July 2000; accepted after revision 27 October 2000.
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ABSTRACT |
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-adrenergic agonist, or isobutylmethyl-xanthine (IBMX, 10-80 µM), a wide spectrum phosphodiesterase (PDE) inhibitor.
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INTRODUCTION |
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The synthesis of nitric oxide (NO) plays an important role in the endothelium-dependent relaxation of various blood vessels. In the heart, the role of NO synthesis is rather controversial (reviewed in Méry et al. 1997; Feron et al. 1999; Kojda & Kottenberg, 1999). NO was soon recognized as an activator of cGMP synthesis in the whole heart, as well as in purified cardiac myocytes. In turn, cGMP co-ordinates the activity of various proteins, such as the cGMP-stimulated phosphodiesterase (PDE2), the cGMP-inhibited-PDE (PDE3) and the cGMP-dependent protein kinase (cG-PK). Accordingly, several NO donors can modulate myocyte shortening, ionic currents and contractile proteins, in a cGMP-dependent manner. More recently, nitrosothiols (SNAP, SNAC, GSNO) or the sydnonymine SIN-1 were found to elicit cGMP-independent effects in cardiac myocytes, including the modulation of calcium channels (Hu et al. 1997; Xu et al. 1998), the inhibition of the creatine kinase (Gross et al. 1996), and the modulation of mitochondrial respiration (Wolin et al. 1997).
While these results tend to support a physiological role of NO in heart muscle, a number of questions remain unanswered. First, in several studies performed on isolated cardiac myocytes, NO donors had no effect on cell shortening (Stein et al. 1993; McDonell et al. 1995, 1997), L-type Ca2+ current (ICa,L) (Thomas et al. 1997), or the cAMP-activated chloride current (Zakharov et al. 1996). Thus, subtle differences between cardiac preparations can blunt the effects of NO at the single cell level. Second, the exact mechanism by which NO or cGMP produces its effect in a given cardiac preparation is not clear. For instance, the inhibitory effect of NO or cGMP on ICa,L in mammalian cardiac myocytes may take place either via activation of PDE2 (Feron et al. 1999) or activation of cG-PK (Méry et al. 1991; Sumii & Sperelakis, 1995; Whaler & Dollinger, 1995). Third, while the effect of cG-PK on ICa,L appears to take place at the level of the L-type Ca2+ channel (or a closely associated protein Méry et al. 1991; Sumii & Sperelakis, 1995), the possibility exists that cG-PK might also act upstream from Ca2+ channel phosphorylation. Indeed, in smooth muscle cells, cG-PK was shown to directly phosphorylate 
i subunits of GTP-binding proteins (G proteins) and/or receptors (Pfeiffer et al. 1995; G.-R. Wang et al. 1998). In addition, cG-PK can increase the spontaneous binding of GTP on subunits and reduce the stimulatory effects of receptor agonists on their GTPase activity (Pfeiffer et al. 1995; Miyamoto et al. 1997; G.-R. Wang et al. 1998).
In the present study, we examined the effects of different NO donors on basal and stimulated ICa,L in ventricular myocytes isolated from rat hearts. In this preparation, we had found previously that cG-PK mediates the inhibitory effect of exogenous cGMP on ICa,L (Méry et al. 1991). While none of the NO donors tested produced any effect on basal ICa,L, the NONOate DEANO produced a profound inhibition of the current stimulated by isoprenaline. We show that cG-PK mediates this effect through the activation of a pertussis toxin-sensitive G protein.
Part of this work has been presented in an abstract form (Abi-Gerges et al. 1997c).
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METHODS |
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The investigation conforms with our institution guidelines which are defined by the European Community guiding principles in the care and use of animals (86/609/CEE, CE Off J no. L358, 18 December, 1986) and the French decree no. 87/848 (J Off République Française, 20 October, 1987, pp. 12245-12248). Authorizations to perform animal experiments according to this decree were obtained from the French Ministère de l'Agriculture et de la Forêt (no. 04226, 12 April, 1991).
Cell isolation and storage
Male Wistar rats (160-250 g) were anaesthetized by intraperitoneal injection of urethane (2 g kg-1) and heparin (2·5 mg kg-1). After all reflex activity had ceased, the animal was killed by opening the chest and rapidly removing the heart. Myocytes were dispersed using collagenase A (0·255 mg ml-1; Boehringer-Mannheim Biochemica, Mannheim, Germany) as previously described (Scamp et al. 1990; Abi-Gerges et al. 1997a).
Electrophysiology
The whole-cell configuration of the patch-clamp technique was used to record the L-type Ca2+ current (ICa,L) on Ca2+-tolerant cells, as described (Scamps et al. 1990; Abi-Gerges et al. 1997a, 1999). The routine protocol consisted of a depolarizing pulse to 0 mV test potential (400 ms duration) elicited every 8 s from a holding potential of -50 mV. The test potential was set at 0 mV because, at this potential, the ICa,L amplitude (Scamps et al. 1990; Méry et al. 1991; Abi-Gerges et al. 1999; see Fig. 2) and steady-state activation are at their maximal values in rat myocytes (Scamps et al. 1990). Current-voltage relationships and inactivation curves were performed as described (Abi-Gerges et al. 1999). The experiments were carried out at room temperature (22-32°C, mean value 25·6 ± 0·1°C, n = 346), and the temperature did not change by >2°C in any given experiment.
During patch-clamp experiments, the maximal amplitude of ICa,L and the current at the end of the 400 ms test potential were measured as described (Abi-Gerges et al. 1997a). Currents were not compensated for capacitive and leak currents. The density of basal ICa,L was 5·25 ± 0·14 pA pF-1 and the density of the current at the end of the 400 ms pulse was 0·37 ± 0·02 pA pF-1 (n = 266). The steady-state value of the end-pulse current was stable over the time course of the experiments (see individual current traces in figures). The effects of the agonists used in this study were not correlated with the amplitude of the end-pulse current (data not shown). The decay of the capacitive transient was fast (< 3 ms), and did not interfere significantly with the activation of the calcium current (mean time to peak 6·2 ± 0·1 ms, n = 266). On-line analysis of the recordings was made possible by programming a PC-compatible 486/50 microcomputer in Assembly language (Borland) to determine, for each membrane depolarization, peak and steady-state current values.
Solutions for patch-clamp recordings
The extracellular solution contained (mM): 107 NaCl, 10 Hepes, 20 CsCl, 4 NaHCO3, 0·8 NaH2PO4, 1·8 MgCl2, 1·8 CaCl2, 5 D-glucose, 5 sodium pyruvate, and 6 × 10-4 tetrodotoxin, pH 7·4 adjusted with CsOH (294 mosmol kg-1). Solutions were superfused onto floating myocytes as described (Abi-Gerges et al. 1997a). The patch pipettes (0·5-1·0 M
) were filled with an intracellular solution containing (mM): 119·8 CsCl, 5 EGTA (acid form), 4 MgCl2, 5 sodium phosphocreatine, 3·1 Na2ATP, 0·42 Na2GTP, 0·062 CaCl2 (pCa 8·5) and 10 Hepes, pH 7·3 adjusted with CsOH (292 mosmol kg-1).
Drugs
3-Morpholino-sydnonimine (SIN-1) was a generous gift from Dr J. Winicki (Hoechst-Houdé Laboratories, Paris-La Défense). Spermine-NONOate (SPNO) and S-nitroso-L-glutathione (GSNO) were from Alexis Corp. (La Jolla, CA, USA); S-nitroso-N-acetyl-d,L-penicillamine (SNAP), 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) from Tocris-Cookson (Bristol, UK) or Alexis Corp.; 8-(4-chlorophenylthio)guanosine-3'-5'-cyclic monophosphorothioate, Rp-isomer (Rp8cG) were from Biolog L.S.I. (Bremen, Germany); 8-(4-chlorophenylthio)-guanosine-3',5'-cyclic monophosphate (8-p-CPT-cGMP) and KT5823 were from Calbiochem-France Biochem (Meudon, France); pertussis toxin (from Bordetella pertussis) was from Sigma-Aldrich (Saint Quentin Fallavier, France); tetrodotoxin was from Latoxan (Rosans, France). All other drugs were from Sigma-Aldrich. Drugs were prepared and used according to manufacturer's instructions. Mock DEANO consisted of a DEANO (100 µM)-containing solution left at room temperature for >20 h. Solutions were prepared by dilution to the desired concentration in the physiological solution at the beginning of each experiment.
Statistical analysis
Results are expressed as means ± S.E.M. Differences between mean values were tested for statistical significance by Student's paired t test, as indicated. In the text, the 'basal' condition for ICa,L refers to the absence of extracellular isoprenaline or IBMX, or intracellular cAMP.
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RESULTS |
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Effect of NO donors on basal ICa,L
NO donors can modulate basal ICa,L in some, but not all cardiac myocytes (Méry et al. 1993; Kirstein et al. 1995; Campbell et al. 1996; Y. G. Wang et al. 1998). In the experiment of Fig. 1A, a rat myocyte was first exposed to control intracellular and extracellular solutions. Under this condition, the amplitude of the basal ICa,L declined slowly (
-5·4 pA min-1), a phenomenon known as 'run-down'. Superfusion of the myocyte with DEANO (100 µM) barely modified the amplitude and the kinetics of the basal ICa,L. As summarized in Fig. 1B, the basal ICa,L amplitude was not significantly modified by different NO donors. These included the NONOate DEANO (100 µM), the nitrosothiols GSNO (1 mM) and SNAP (1 mM), the ferrocyanate SNP (500 µM), and the sydnonymine SIN-1 (1 mM). Lower concentrations of these compounds (down to 1 µM) also had no effect on basal ICa,L (data not shown).
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A, a myocyte was first exposed to control intracellular and extracellular control solutions. ICa,L ( | ||
Since nitrosylation and/or oxidation might account for effects of NO donors on the basal ICa,L in other preparations (Campbell et al. 1996; Hu et al. 1997) we further investigated the sensitivity of basal ICa,L in rat myocytes to the effects of reducing or oxidizing agents. The basal ICa,L was not changed when the myocyte was superfused with 1 mM reduced glutathione (5·9 ± 2·6 % over basal, n = 5), 1 mM dl-dithiothreitol (dl-DTT) (2·3 ± 1·6 % over basal, n = 6), 0·1 mM N-acetyl-penicillamine (-1·6 ± 0·9 % over basal, n = 4). In contrast, extracellular application of a superoxide anion generator LY83583 (10 µM) inhibited the basal ICa,L (-36·9 ± 2·5 % over basal, n = 17, P < 0·001). Therefore, the basal activity of L-type Ca2+ channels in rat myocytes was sensitive to an oxidative treatment but not to NO donors.
Inhibitory effect of DEANO on the -adrenergic stimulation of ICa,L
NO donors can reduce the -adrenergic stimulation of ICa,L in frog and guinea-pig ventricular myocytes (Méry et al. 1993; Levi et al. 1994; Whaler & Dollinger, 1995; reviewed in Méry et al. 1997). We first investigated the effects of DEANO in the presence of isoprenaline (Iso), a non-selective -adrenergic agonist. In the experiment of Fig. 2A, a myocyte was exposed to control solutions and the ICa,L amplitude reached a steady state 11·8 min after the beginning of the recording of ICa,L. The myocyte was then superfused with 10 nM Iso, which induced a 2-fold increase in ICa,L. In the continuing presence of Iso, superfusion with 100 µM DEANO elicited a pronounced reduction in the response of ICa,L to Iso. The inhibitory effect of DEANO was reversible. Figure 2B shows the current-voltage relationships of ICa,L (filled symbols) and of the steady-state current measured at the end (400 ms) of the test pulse (open symbols) in the same experiment as in Fig. 2A. The effects of Iso and DEANO on ICa,L were homogeneous across the voltage range and neither drug produced a change in the steady-state current (similar findings were obtained in 10 other experiments). In addition, DEANO did not change the inactivation curve of ICa,L (data not shown). Thus, DEANO produced an anti-adrenergic effect on ICa,L which occurred in a voltage-independent manner.
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-adrenergic stimulation of ICa,L in rat ventricular myocytesA, a myocyte was first exposed to control intracellular and extracellular solutions. Applications of isoprenaline (Iso, 10 nM) and DEANO (100 µM) are indicated by the horizontal lines. Current traces on top were recorded at the times indicated by the corresponding letters on the main graph. The dotted line indicates the zero-current level. B (same experiment as in A), current-voltage relationships of ICa,L (filled symbols) and of the steady-state current at the end of the pulse (open symbols) obtained under basal conditions, and in the presence of Iso (10 nM) with or without DEANO (100 µM). | ||
The inhibitory effect of DEANO (100 µM) was studied at three different Iso concentrations (1, 3 and 10 nM). As summarized in Fig. 3, increasing the Iso concentration progressively reduced the inhibitory effect of DEANO on ICa,L suggesting that the inhibitory effect of DEANO was somewhat competitive with the stimulatory effect of Iso. The involvement of NO in the anti-adrenergic effect of DEANO was investigated using mock DEANO (see Methods). The Iso (1 nM) stimulation of ICa,L was identical in the absence and in the presence of 100 µM mock DEANO (176·8 ± 11·9 % and 188·9 ± 10·1 % of control, respectively, n = 4). In addition, the stimulatory effect of 1 nM Iso on ICa,L was not changed in the presence of 100 µM diethylamine (179·1 ± 16·0 % and 177·0 ± 20·0 % of control, respectively, n = 4), the other metabolite of DEANO. Thus, the inhibitory effect of DEANO on Iso-stimulated ICa,L was mediated by NO and not by one of its metabolites.
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-adrenergic stimulation of ICa,L in rat ventricular myocytes
The amplitude of ICa,L in the presence of Iso (1, 3 or 10 nM), without ( | ||
Effect of other NO donors on the -adrenergic stimulation of ICa,L
For a comparison with the effect of DEANO, we examined the effects of other NO donors on the Iso-stimulated ICa,L, namely SPNO (a NONOate like DEANO), SIN-1 (a sydnonymine) and SNAP (a nitrosothiol). Figure 4A shows a typical experiment in which SIN-1 (100 µM) had no effect on ICa,L stimulated by 10 nM Iso. As summarized in Fig. 4B, SIN-1 (100 µM) as well as the two other NO donors, SPNO (100 µM) and SNAP (100 µM), produced inconsistent and non-significant effects on ICa,L stimulated with either 1 or 10 nM Iso. In addition, the shape of the current-voltage relationship of the Iso-stimulated ICa,L was not modified in the presence of SIN-1, SNAP, or SPNO (n = 8, 5 and 2, respectively, data not shown). These negative results were not due to the wash-out of some cellular component in the whole-cell configuration of the patch-clamp technique since SNAP also failed to modify the Iso-stimulated ICa,L when tested in the perforated-patch configuration (data not shown, see also Thomas et al. 1997). They were not due either to a difference in temperature, since the mean temperatures of the experiments were similar with DEANO (25·3 ± 0·3°C, n = 25), SPNO (28·0 ± 0·5°C, n = 5), SIN-1 (28·5 ± 0·5°C, n = 3) and SNAP (28·3 ± 0·4°C, n = 4) when tested on top of 1 nM Iso. Thus, among a total of four NO donors tested, DEANO was the only one to consistently produce an inhibitory effect on Iso-stimulated ICa,L in rat ventricular myocytes (see Maragos et al. 1991; Feelisch et al. 1991).
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-adrenergic stimulation of ICa,L in rat ventricular myocytes
A, a myocyte was first exposed to control intracellular and extracellular solutions. Applications of Iso (10 nM) and SIN-1 (100 µM) are indicated by the horizontal lines. Current traces on top were recorded at the times indicated by the corresponding letters on the main graph. The dotted line indicates the zero-current level. B, summary of the effects of three NO donors (SNAP, SIN-1, SPNO, at 100 µM) on ICa,L in the presence of either 1 nM ( | ||
Contribution of guanylyl cyclase to the anti-adrenergic effect of DEANO on ICa,L
To evaluate the contribution of cGMP to the modulation of ICa,L by DEANO, we examined the effect of DEANO in the presence of ODQ, an inhibitor of the NO-sensitive guanylyl cyclase (Kojda et al. 1996; Abi-Gerges et al. 1997b). In the typical experiment of Fig. 5A, a myocyte was exposed to control solutions, and then superfused with 3 nM Iso. The stimulatory effect of the -adrenergic agonist was reduced by about 50 % upon addition of DEANO (100 µM). In the continuing presence of Iso plus DEANO, superfusion with ODQ (10 µM) increased ICa,L, eliminating most of the inhibitory effect of DEANO. The effect of ODQ was slowly reversible upon wash-out of the drug. On average (Fig. 5B), ODQ (10 µM) fully antagonized the inhibitory effects of DEANO on ICa,L, in the presence of 1, 3 or 10 nM Iso. However, ODQ (10 µM) did not modify Iso (10 nM)-stimulated ICa,L in the absence of DEANO (-2·2 ± 2·1 % over Iso level, n = 4). Thus, these results indicate that the inhibitory effect of DEANO on Iso-stimulated ICa,L in rat ventricular myocytes is mediated by activation of the NO-sensitive guanylyl cyclase.
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-adrenergic stimulation of ICa,L in rat ventricular myocytes
A, a myocyte was first exposed to control intracellular and extracellular solutions. Applications of Iso (3 nM), DEANO (100 µM) and ODQ (10 µM) are indicated by the horizontal lines. Current traces on top were recorded at the times indicated by the corresponding letters on the main graph. The dotted line indicates the zero-current level. B, summary of the effects of DEANO without ( | ||
Contribution of cG-PK to the anti-adrenergic effect of DEANO on ICa,L
We have shown previously that an intracellular application of cGMP in rat ventricular myocytes produces an inhibition of cAMP-stimulated ICa,L that involves the activation of cG-PK (Méry et al. 1991). Similarly, the Iso (1 nM) stimulation of ICa,L was strongly reduced in the presence of 8-p-CPT-cGMP (100 µM), a selective activator of cG-PK (from 235·1 ± 8·9 % to 168·2 ± 6·9 % stimulation, n = 4, P < 0·005). Next, the role of the cG-PK in the anti-adrenergic effect of DEANO was examined using (Rp)-8-CPT-cGMP (Rp8cG), a membrane-permeant inactive analogue of cGMP which competes with cGMP for the binding on cG-PK (Butt et al. 1994). In the typical experiment of Fig. 6A, the -adrenergic stimulation of ICa,L (with 1 nM Iso) was strongly inhibited by DEANO (100 µM). This anti-adrenergic effect of DEANO was fully antagonized by superfusion of the cell with 10 µM Rp8cG, in a reversible manner. Figure 6B summarizes the results of several similar experiments. At 1 and 10 nM Iso, Rp8cG (10 µM) fully reversed the inhibitory effect of DEANO (100 µM) on ICa,L. We also examined the effects of KT5823, another cG-PK inhibitor structurally unrelated to cGMP (Komalavila & Lincoln, 1996). As summarized in Fig. 6B, KT5823 (0·1 or 0·3 µM) also fully reversed the anti-adrenergic effect of DEANO (100 µM). In the absence of DEANO, neither Rp8cG (10 µM) nor KT5823 (0·1-0·3 µM) had any significant effect on Iso-stimulated ICa,L (data not shown). Thus, these results demonstrate that cG-PK mediates the inhibitory effect of DEANO on ICa,L in rat ventricular myocytes.
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-adrenergic stimulation of ICa,L in rat ventricular myocytes
A, a myocyte was first exposed to control intracellular and extracellular solutions. Applications of Iso (1 nM), DEANO (100 µM) and Rp8cG (10 µM) are indicated by the horizontal lines. Current traces on top were recorded at the times indicated by the corresponding letters on the main graph. The dotted line indicates the zero-current level. B, summary of the effects of DEANO (100 µM) without ( | ||
Effect of DEANO on cAMP-stimulated ICa,L in rat ventricular myocytes
When elicited by intracellular dialysis of cGMP in rat ventricular myocytes, cG-PK-dependent inhibition of ICa,L appeared to take place at the level of the Ca2+ channels (Méry et al. 1991; Sumii & Sperelakis, 1995). In other cell types, cG-PK can modulate the initial steps of signal transduction, at the level of receptors and/or G proteins (Pfeiffer et al. 1995; Miyamoto et al. 1997; G.-R. Wang et al. 1998). To discriminate between these possibilities, we first studied the effect of DEANO in myocytes dialysed with cAMP through the patch pipette, in order to bypass the steps involved in cAMP production. In the experiment of Fig. 7A, the patch pipette was filled with a control intracellular solution supplemented with 30 µM cAMP, and intracellular dialysis of the myocyte started when the membrane patch was ruptured. The diffusion of cAMP into the cytosol induced a large increase in ICa,L (to 44·9 pA pF-1). Surprisingly, superfusion of the myocyte with DEANO (100 µM) did not modify the cAMP-stimulated ICa,L. The effect of another NO donor, SNP, is shown in Fig. 7B. In this experiment, the myocyte was first exposed to control intracellular and extracellular solutions. Intracellular dialysis with cAMP (10 µM) started at the time indicated by the arrow, inducing a 2·5-fold increase in ICa,L (to 26·7 pA pF-1). Superfusion of the myocyte with increasing concentrations of SNP (5, 50 and 500 µM) had no effect on the cAMP-stimulated ICa,L.
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A, the pipette solution contained 30 µM cAMP, and cAMP dialysis started when the patch was ruptured (arrow). B, a myocyte was first dialysed with the control solution, and intracellular dialysis with 30 µM cAMP started at the time indicated by the arrow. Applications of DEANO (100 µM) in A or SNP (5, 50 and 500 µM) in B were performed as indicated by the horizontal lines. In A, current traces on top were recorded at the times indicated by the corresponding letters on the main graph. The dotted line indicates the zero-current level. C, summary of the effects of DEANO, SNP and SNAP on the cAMP (10-100 µM)-stimulated ICa,L. Variations are given as percentage change over the amplitude of the cAMP-stimulated ICa,L. Bars are the means and lines are the S.E.M. of the number of experiments indicated near the bars. | ||
The effects of DEANO, SNP and SNAP on ICa,L in cAMP (10-100 µM)-dialysed myocytes are summarized in Fig. 7C. On average, the density of ICa,L was elevated to 23·02 ± 3·57 pA pF-1 (n = 12). None of the NO donors significantly changed the cAMP-stimulated ICa,L. Thus, since DEANO inhibits the Iso-stimulated ICa,L but not the cAMP-stimulated ICa,L, it must act at a step located upstream from cAMP production in the cAMP signalling cascade leading to ICa,L stimulation. In the following, we tested the hypothesis that DEANO via activation of cG-PK interferes with the receptor-dependent modulation of adenylyl cyclase.
Effect of DEANO on Iso-stimulated ICa,L in pertussis toxin-treated myocytes
In vitro experiments demonstrated that cG-PK could modulate the activity of Gi proteins, which are negatively coupled to adenylyl cyclase in rat cardiac myocytes (Pfeiffer et al. 1995; Miyamoto et al. 1997). Therefore, we studied the effect of DEANO in pertussis toxin (PTX)-treated myocytes, where Gi/Go proteins are irreversibly inactivated. Myocytes were incubated for 4-6 h (before patch-clamp experiments) in the presence of 0·5 µg ml-1 PTX, at 37°C (Hilal-Dandan et al. 1992). In the typical experiment shown in Fig. 8A, Iso (1 nM) was applied to a PTX-treated myocyte, inducing a fast and large rise in ICa,L. However, under these conditions, superfusion of the myocyte with DEANO (100 µM) had no effect on ICa,L. Since the muscarinic receptor agonist acetylcholine (ACh) produces an anti-adrenergic effect on ICa,L which clearly involves activation of PTX-sensitive G proteins (reviewed in Méry et al. 1997; Feron et al. 1999), we tested the efficacy of PTX to inactivate Gi/Go proteins by investigating the effect of a subsequent application of ACh (1 µM) on Iso-stimulated ICa,L. As shown in Fig. 8A, and summarized in Fig. 8B, PTX treatment fully abrogated the anti-adrenergic effect of both ACh (1 µM) and DEANO (100 µM). Note that when normalized to the Iso-stimulated ICa,L amplitude, ACh, but not DEANO, tended to potentiate the effect of the -adrenergic agonist in PTX-treated myocytes (Fig. 8C).
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-adrenergic stimulation of ICa,L in PTX-treated rat ventricular myocytesA, a myocyte was incubated with pertussis toxin (PTX, 0·5 µg ml-1, 4 h, 37 °C) prior to the experiment. It was first exposed to control extracellular and intracellular solutions, and applications of Iso (1 nM), DEANO (100 µM) and acetylcholine (ACh, 1 µM) were performed as indicated by the horizontal lines. Current traces on top were recorded at the times indicated by the corresponding letters on the main graph. The dotted line indicates the zero-current level. B and C, summary of the effects of DEANO (100 µM) and ACh (1 µM) on the Iso (0·01 and 1 nM)-stimulated ICa,L in PTX-treated myocytes. The amplitude of ICa,L is presented as percentage increase over basal amplitude (in B) or as percentage variations from the amplitude of the Iso-stimulated ICa,L (in C). Bars are the means and lines are the S.E.M. of the number of experiments indicated near the bars. Significant differences from Iso level are indicated as: * P < 0·05. | ||
We next examined whether the effect of PTX was taking place upstream or downstream from cG-PK activation. For this, we investigated the effect 8-p-CPT-cGMP (100 µM) in PTX-treated myocytes. In four such cells, the stimulation of ICa,L by Iso 1 nM was only slightly attenuated by 8-p-CPT-cGMP (from 192·0 ± 11·0 % to 171·7 ± 11·0 % of control, P = 0·1), demonstrating that PTX treatment blunted the inhibitory effect of a direct activation of the cG-PK on ICa,L. In these cells, the Iso-stimulated ICa,L was also not affected by ACh (1 µM, 198·9 ± 8·0 % of control). Altogether, these data demonstrate that DEANO inhibits ICa,L via a cG-PK modulation of a PTX-sensitive Gi/Go protein.
Effect of DEANO on IBMX-stimulated ICa,L in pertussis toxin-treated myocytes
While PTX treatment of rat ventricular myocytes did not modify basal ICa,L density (5·2 ± 0·4 pA pF-1, n = 23, in PTX vs. 5·3 ± 0·1 pA pF-1, n = 266, in control), it somewhat increased the Iso response (at 0·01 and 1 nM, compare Fig. 8B and Fig. 3). Thus, the anti-adrenergic effect of DEANO might have been blunted in PTX-treated myocytes as a result of a saturating production of cAMP. Therefore, we re-examined the involvement of Gi/Go proteins in the modulation of ICa,L by DEANO under conditions where intracellular cAMP was non-maximally increased. To do this, we used low concentrations of isobutyl-methyl-xanthine (IBMX), a broad spectrum phosphodiesterase inhibitor which, unlike Iso, does not activate cAMP synthesis but increases cAMP levels by reducing its degradation. Figure 9A shows a typical experiment performed in a control (untreated) myocyte. IBMX (40 µM) elicited a 25 % increase in basal ICa,L and this effect was totally abolished by further application of DEANO (100 µM). This effect of DEANO clearly involved NO generation, since the addition of the NO scavenger carboxy PTIO (Akaike et al. 1993) totally antagonized the effect of the NO donor. Indeed, in three myocytes where the stimulation of ICa,L by 40 µM IBMX (154·2 ± 4·9 % of control) had been reduced by 100 µM DEANO (to 118·5 ± 2·9 % of control, P < 0·005), the addition of carboxy PTIO (100 µM) to the DEANO solution restored the stimulation of ICa,L to its level in Iso alone (to 156·8 ± 1·7 % of control).
In contrast to control cells, DEANO failed to inhibit the response of ICa,L to IBMX in PTX-treated myocytes. In the typical experiment of Fig. 9B, neither DEANO (100 µM) nor ACh (1 µM) produced any inhibition of ICa,L stimulated by IBMX (20 µM). The results of several similar experiments are summarized in Fig. 9C. On average, IBMX (10-80 µM) exerted submaximal stimulatory effects on ICa,L. While ACh (1 µM) strongly reduced the IBMX response in control (untreated) myocytes, this inhibition was absent in PTX-treated myocytes indicating that Gi/Go proteins were efficiently blocked by PTX. Similarly, DEANO (100 µM) strongly antagonized the IBMX-stimulated ICa,L in untreated myocytes, and the effect was abolished in PTX-treated myocytes. Thus, the inhibitory effect of DEANO on ICa,L shared some similarity with the effect of ACh, in that both effects required the integrity of PTX-sensitive G proteins. Note that in one out of the nine experiments in PTX-treated myocytes, ACh had no effect on IBMX-stimulated ICa,L while DEANO still elicited an inhibitory effect.
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Untreated (A) and PTX-treated (B) myocytes were first exposed to control extracellular and intracellular solutions. Applications of IBMX (40 µM in A, 20 µM in B), DEANO (100 µM, in A and B) and ACh (1 µM, in B) are indicated by the horizontal lines. Current traces on top were recorded at the times indicated by the corresponding letters on the main graphs. The dotted lines indicate the zero-current level. C, summary of the effects of IBMX (10-80 µM) on ICa,L, used either alone ( | ||
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DISCUSSION |
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In this study, we report that ICa,L is regulated by NO in rat ventricular cardiac myocytes. The NO donor DEANO exerted a pronounced anti-adrenergic effect, which involved the activation of the NO-sensitive guanylyl cyclase and cG-PK. The main locus of action of cG-PK appeared to be a PTX-sensitive G protein.
In agreement with studies in frog and guinea-pig ventricular myocytes (Méry et al. 1993; Levi et al. 1994; Whaler & Dollinger, 1995), we found that a NO donor, DEANO, strongly inhibited the -adrenergic stimulation of ICa,L in rat ventricular myocytes. The inhibitory effect of DEANO occurred in the micromolar range of concentrations and was not mimicked by mock DEANO or diethylamine. This effect clearly involved NO generation, since the addition of the NO-scavenger carboxy PTIO (Akaike et al. 1993) totally antagonized the effect of the NO donor. In contrast, other NO donors (SIN-1, SNAP and SPNO, a NONOate like DEANO) did not significantly modify the Iso-stimulated ICa,L. However, the rate of NO release by these NO donors is slow compared with that of DEANO (Feelisch et al. 1991; Maragos et al. 1991; Ferrero et al. 1999). As suggested by Kojda et al. (1996), the time course of NO release appears to be critical when comparing the functional effects of different NO donors. Nevertheless, although DEANO is 1000-fold more potent than SIN-1, SNP or SNAP, all these NO donors inhibit the Iso-stimulated ICa,L in frog ventricular myocytes (e.g. see Méry et al. 1993; Abi-Gerges et al. 1997b). A possible explanation for this discrepancy may come from the observation that rat and mouse ventricles exhibit higher myoglobin contents than other species (O'Brien et al. 1992). Since myoglobin is known as a potent NO scavenger, it may hinder the effects of slow NO sources, such as SIN-1, SNAP and SPNO, but not that of a fast NO source like DEANO ( Ishibashi et al. 1992; Beckman & Koppenol, 1996).
Another interesting observation was that DEANO behaved as an apparent competitive antagonist of the response of ICa,L to Iso in rat myocytes. We found that the inhibitory effect of DEANO on ICa,L was reduced as the concentration of Iso was increased from 1 to 10 nM. This observation might help to explain the discrepancy between negative and positive results obtained in the literature when comparing the efficacy of a given NO donor in different cardiac preparations (Thomas et al. 1997).
DEANO was shown to enhance cGMP production in isolated rat ventricular myocytes (Kojda et al. 1996). In agreement with this finding, we found that activation of the cGMP pathway accounted for the anti-adrenergic effect of DEANO on ICa,L in the same preparation. Indeed, the effect of DEANO on ICa,L was antagonized by ODQ, a specific guanylyl cyclase inhibitor (Abi-Gerges et al. 1997b; Sandirasegarane & Diamond, 1999). Moreover, the effect of DEANO on ICa,L was mimicked by 8-p-CPT-cGMP, a cG-PK activator, and antagonized by Rp8cG and KT5823, two cG-PK inhibitors (Butt et al. 1994; Komalavila & Lincoln, 1996). Therefore, the endogenous activation of the cG-PK accounted for the anti-adrenergic effect of DEANO on ICa,L.
Earlier studies in rat ventricular myocytes have shown that cG-PK was involved in the inhibitory effects of exogenous cGMP on ICa,L (Méry et al. 1991; Sumii & Sperelakis, 1995). Intracellular dialysis of isolated myocytes with cGMP or with constitutively active cG-PK elicited inhibitory effects on ICa,L when the current was maximally stimulated with 100 µM IBMX or 100 µM intracellular cAMP. In addition, NO donors inhibited IBMX- or cAMP-stimulated ICa,L in guinea-pig myocytes (Levi et al. 1994; Whaler & Dollinger, 1995). Accordingly, L-type Ca2+ channels were viewed as a major target of cG-PK in these cells (Méry et al. 1991; Levi et al. 1994; Whaler & Dollinger, 1995; Sumii & Sperelakis, 1995).
However, we found here that in rat ventricular myocytes the inhibitory effect of DEANO on ICa,L was strongly reduced when the concentration of Iso was increased to 10 nM (Fig. 3) or in the presence of 200 µM IBMX (data not shown). Moreover, DEANO did not inhibit the stimulation of ICa,L induced by intracellular cAMP. Therefore, the major target of cG-PK may differ when activated by endogenous cGMP production (this study) or by intracellular dialysis of exogenous cGMP (Méry et al. 1991). Our present results suggest that cG-PK acts at the level of G proteins when guanylyl cyclase is stimulated by NO since the inhibitory effect of DEANO on ICa,L was antagonized by PTX, which inactivates Gi/Go proteins. Both Gi and Go proteins are involved in the regulation of ICa,L (Méry et al. 1997; Valenzuala et al. 1997; Feron et al. 1999), but only Gi-dependent pathways were shown to be modulated by cG-PK (Pfeiffer et al. 1995). Activation of cG-PK was shown to phosphorylate i subunits of G proteins (Pfeiffer et al. 1995) and to increase the spontaneous binding of GTP (Miyamoto et al. 1997). Our results strongly suggest that this modulation of i subunits of Gi proteins accounts for the observed cG-PK-dependent, PTX-sensitive inhibitory effect of DEANO on Iso-stimulated ICa,L in rat myocytes. Yet, we can only speculate on the contribution of this mechanism to the regulation of the Iso-stimulated ICa,L by NO in guinea-pig myocytes (Levi et al. 1994; Whaler & Dollinger, 1995). Interestingly, rat ventricles express higher levels of myoglobin than guinea-pig ventricles (O'Brien et al. 1992), and high myoglobin content blunted cGMP production induced by NO (Ishibashi et al. 1992). Therefore, we propose that only high levels of cGMP can elicit the direct inhibition of L-type calcium channels by cG-PK (as seen during dialysis of cGMP, or in guinea-pig myocytes), while moderate levels of cGMP induce the specific regulation of Gi proteins by cG-PK (as seen in rat myocytes).
In addition to the regulation of cGMP production, NO can regulate directly the activity of several proteins in the heart, including the L-type Ca2+ channels. Nitrosothiols and SIN-1 enhanced ICa,L in a cGMP-independent manner in ferret ventricular myocytes (Campbell et al. 1996), but nitrosothiols inhibited the activity of cloned L-type Ca2+ channels in HEK293 cells (a human embryonic kidney cell line) (Hu et al. 1997). In rat ventricular myocytes, as in cardiac myocytes from other species and tissues (Méry et al. 1993; Levi et al. 1994; Kirstein et al. 1995; Wahler & Dollinger, 1995; Y. G. Wang et al. 1998; Feron et al. 1999), NO donors (including nitrosothiols and SIN-1) did not elicit a cGMP-independent effect on ICa,L. Interestingly, we found that the basal ICa,L in rat ventricular myocytes was not modified by reducing agents but was inhibited by LY83583, a superoxide anion generator. Hu et al. (1997) also reported that the basal activity of the L-type Ca2+ channel was not changed by reducing agents but was inhibited by oxidant treatments. In contrast, ICa,L was inhibited by reducing agents (glutathione, dl-DTT) and enhanced by an oxidant (DTNB) in ferret ventricular myocytes (Campbell et al. 1996). Altogether, these data suggest that the different effects of NO donors on the basal ICa,L might require different redox environments. In addition, it is possible that the 'direct' effects of NO donors do not take place on the L-type Ca2+ channel itself, but on other auxiliary proteins and/or on lipids. While the reason for these discrepancies remains to be fully elucidated, our results clearly demonstrate that NO can recruit an endogenous cG-PK in rat ventricular myocytes, leading to a tight control of the -adrenergic stimulation of L-type Ca2+ channel activity.
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We thank Patrick Lechêne for skilful technical assistance, Florence Lefebvre for preparation of the cells, Drs Michel Chesnais, Bernd K. Fleischmann and Renzo Levi for helpful discussions. We are grateful to Dr Xinqiang Han for sharing unpublished results. N. Abi-Gerges was supported by the Fondation pour la Recherche Medicale (F.R.M.) and the Association Française contre les Myopathies (A.F.M.).
Corresponding author
N. Abi-Gerges: INSERM U-446, Laboratoire de Cardiologie Cellulaire et Moléculaire, Université Paris-Sud, Faculté de Pharmacie, 5 rue Jean-Baptiste Clément, F-92296 Châtenay-Malabry Cedex, France.
Email: naja.abi-g{at}cep.u-psud.fr
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) was elicited at 0 mV from a holding potential of -50 mV. Superfusion of the myocyte with 100 µM DEANO is indicated by the horizontal line. Current traces on top were recorded at the times indicated by the corresponding letters on the main graph. The dotted line indicates the zero-current level. B, summary of the effects of DEANO (100 µM), GSNO (1 mM), SNAP (1 mM), SNP (500 µM) and SIN-1 (1 mM) on basal ICa,L amplitude. The amplitude of ICa,L in the presence of NO donors was normalized to the amplitude of basal ICa,L in control conditions (set to 100 %). Bars are the means and lines are the S.E.M. of the number of experiments indicated near the bars.
) or with DEANO (100 µM, 
), was normalized to the basal ICa,L amplitude (set to 100 %). Bars are the means and lines are the S.E.M. of the number of experiments indicated near the bars. Significant differences from basal (*) or Iso levels (
) are indicated as: *P < 0·001; **,