Local response of L-type Ca2+ current to nitric oxide in frog ventricular myocytes

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Local response of L-type Ca²⁺ current to nitric oxide in frog ventricular myocytes

Michael Dittrich †, Jonas Jurevicius , Marie Georget , Francesca Rochais , Bernd Kurt Fleischmann †, Jürgen Hescheler † and Rodolphe Fischmeister *

* Laboratoire de Cardiologie Cellulaire et Moléculaire, INSERM U-446, Université de Paris-Sud, Faculté de Pharmacie, F-92296 Châtenay-Malabry, France and † Institut für Neurophysiologie, Universität zu Köln, Robert-Koch-Straße 39, D-50931 Cologne, Germany

MS 11456 Received 26 July 2000; accepted after revision 5 March 2001

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

The regulation of L-type Ca²⁺ current (I_Ca) by the two nitric oxide (NO) donors sodium nitroprusside (SNP, 1 µM to 1 mM) and (±)-S-nitroso-N-acetylpenicillamine (SNAP, 3 or 10 µM) was investigated in frog ventricular myocytes using double voltage clamp and double-barrelled microperfusion techniques.
SNP and SNAP depressed the isoprenaline (ISO, 10-100 nM)- or forskolin (FSK, 1 µM)-mediated stimulation of I_Ca via cGMP activation of the cGMP-stimulated phosphodiesterase (PDE2). Complete inhibition of the ISO (100 nM) response was observed at 1 mM SNP.
When SNP was applied locally, i.e. to one-half of the cell, and ISO to the whole cell, the response of I_Ca to ISO was strongly antagonized in the cell half exposed to SNP (up to 100 % inhibition at 1 mM SNP) but a relatively small depression was observed in the other half of the cell (only 20 % inhibition at 1 mM SNP).
The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO, 1 mM) reversed the local effect of SNAP (3 µM) on FSK-stimulated I_Ca when applied to the same side as the NO donor, but had no effect when applied to the other side of the cell.
A local application of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA, 30 µM), a selective inhibitor of PDE2, fully reversed the local effect of SNP (100 µM) or SNAP (10 µM) on I_Ca but had no effect on the distant response.
When EHNA was applied on the distant side, with SNP (1 mM) and ISO (100 nM) applied locally, the distant effect of SNP was fully reversed.
Our results demonstrate that in frog ventricular myocytes stimulation of guanylyl cyclase by NO leads to a strong local depletion of cAMP near the L-type Ca²⁺ channels due to activation of PDE2, but only to a modest reduction of cAMP in the rest of the cell. This may be explained by the existence of a tight microdomain between L-type Ca²⁺ channels and PDE2.

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

Regulation of the cardiac L-type Ca²⁺ channel current (I_Ca) plays a determinant role in the control of heart function by hormones and neurotransmitters (for reviews see Hartzell, 1988; McDonald et al. 1994; Hove-Madsen et al. 1996; Shimoni, 1999). In particular, stimulation of I_Ca is an initial event in the positive inotropic effect produced by activation of the sympathetic nervous system. This well-documented phenomenon occurs by liberation of noradrenaline at the sympathetic nerve terminals acting at myocardial $beta$ -adrenergic receptors (Brodde & Michel, 1999), hence activating the cAMP cascade and leading to phosphorylation by cAMP-dependent protein kinase (PKA) of a multitude of regulatory proteins; these include the L-type Ca²⁺ channel (VDCC) or a closely associated protein (Osterrieder et al. 1982; for reviews see Hartzell, 1988; McDonald et al. 1994; Striessnig, 1999), phospholamban (Simmerman & Jones, 1998), the sarcoplasmic reticulum Ca²⁺ release channel (Hain et al. 1995; Valdivia et al. 1995) and contractile proteins (Rapundalo, 1998). While this signalling cascade has been elucidated over the last decade, recent interest has focused on whether the various signalling components involved are co-localized in microdomains. We have recently shown, using double voltage clamp and double-barrelled perfusion techniques, that in frog ventricular myocytes I_Ca is regulated in a cytoplasmic cAMP compartment positioned in close vicinity to the cell membrane (Jurevicius & Fischmeister, 1996). Moreover, this compartmentation of cAMP is provided by the tight spatial control of phosphodiesterases (PDEs) which prevent diffusion over the length of the cardiomyocyte (Jurevicius & Fischmeister, 1996). Positioning of A-kinase anchor proteins in the close proximity of the VDCC may further enhance the local control of I_Ca by cAMP-dependent processes (Gao et al. 1997).

Besides the well-studied cAMP-mediated regulation of I_Ca, recent studies have focused on the nitric oxide (NO)-mediated regulation of I_Ca. While some authors suggest a direct, though controversial, modulation of I_Ca by NO (Campbell et al. 1996; Hu et al. 1997), a large number of studies demonstrate that NO modulates I_Ca through activation of soluble guanylyl cyclase and accumulation of intracellular cGMP (Méry et al. 1993; Levi et al. 1994; Kirstein et al. 1995; Wahler & Dollinger, 1995; Wang et al. 1998; Feron et al. 1999). This cyclic nucleotide may exert opposite effects on I_Ca, depending both on the amount of cGMP produced and on the complex interplay of three putative cGMP targets: cGMP-dependent protein kinase (PKG) (Méry et al. 1991; Sumii & Sperelakis, 1995), cGMP-stimulated phosphodiesterase (PDE2) (Hartzell & Fischmeister, 1987; Méry et al. 1995; Han et al. 1998; Feron et al. 1999; Ji et al. 1999) and cGMP-inhibited phosphodiesterase (PDE3) (Ono & Trautwein, 1991; Méry et al. 1993; Kirstein et al. 1995; Wang et al. 1998). For instance, inhibition of PDE3, which produces an increase in I_Ca, may occur at lower cGMP concentrations than activation of PDE2, which produces a decrease in I_Ca (Méry et al. 1993; Kirstein et al. 1995).

In the frog heart, inhibition of PDE3 or activation of PKG contributes little or nothing to the overall effect of NO donors on I_Ca (Lohmann et al. 1991). On the contrary, a large body of evidence exists in favour of a PDE2-mediated inhibition of I_Ca by cGMP and/or NO donors in this preparation (Fischmeister & Hartzell, 1987; Méry et al. 1993, 1995). This inhibition occurs only when I_Ca has been first stimulated by activation of the cAMP cascade, whether this has been done by activation of the $beta$ -adrenergic receptors with isoprenaline (ISO), direct activation of adenylyl cyclase (AC) with forskolin (FSK), or direct elevation of cAMP levels by intracellular dialysis of the cell. The prevalent role of PDE2 in this preparation is further supported by the finding that, unlike in mammalian ventricular tissue, PDE2 represents the dominant PDE isoform present in the membrane fraction of frog ventricles (Simmons & Hartzell, 1988; Lugnier et al. 1992). The preferential localization of PDE2 at the membrane may suggest that this enzyme, when activated by an elevation of cGMP, will contribute to a depletion of cAMP at the intracellular face of the sarcolemmal membrane and thus modify the spatial distribution of cAMP inside the cell. However, for technical reasons, this hypothesis has never been directly tested in an intact myocyte.

In the present study, our aim was to gain insight into the interaction of the NO/cGMP and cAMP pathways participating in the regulation of I_Ca in frog ventricular myocytes, with a particular emphasis on the spatial distribution of the signalling molecules involved. For this purpose we have used a method recently established by our group, which allows separate recording of membrane currents from and application of substances to the two halves of a cardiomyocyte (Jurevicius & Fischmeister, 1996). This technique is based on the use of double voltage clamp with a pipette attached to either end of the myocyte, and a double micro-barrelled perfusion system sealed by means of a diaphragm pushed against the middle of the cell. Since the amplitude of peak I_Ca is an estimate of the cAMP concentration near the VDCC (Fischmeister & Hartzell, 1990; Hartzell et al. 1991), the effect of the NO donors SNP and SNAP on I_Ca can be extrapolated as a reliable estimate for submembrane cAMP levels. The relative amplitude of I_Ca in the two halves of the cell is a measure of a possible cAMP gradient along the cell. We provide evidence that NO-mediated signalling remains in the local environment and is co-associated closely with the local cAMP concentration, implying the formation of signalling microdomains.

A preliminary report of these results has appeared elsewhere (Dittrich et al. 1999).

METHODS

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Abstract
Introduction
Methods
Results
Discussion
References

The investigation conforms with our institutional guidelines which are defined by the European Community Guiding Principles in the Care and Use of Animals, and French decree no 87/848 of October 19, 1987. Authorization to perform animal experiments according to this decree was obtained from the French Ministère de l'Agriculture, de la Pêche et de l'Alimentation (no. 7475, May 27, 1997).

Patch-clamp recordings with frog ventricular myocytes

Ventricular myocytes were enzymatically isolated from frog (Rana esculenta) hearts with a combination of collagenase (Yakult, Tokyo, Japan) and trypsin (Sigma, St Louis, USA) as described (Fischmeister & Hartzell, 1986). Briefly, frogs were killed by decapitation and double pithing. The isolated cells were stored at 4 °C until use (2-72 h after dissociation). With the exception of the experiments illustrated in Fig. 1, which were carried out using standard single electrode whole-cell recording (Fischmeister & Hartzell, 1986), rod-shaped Ca²⁺-tolerant frog ventricular myocytes (200-400 µm long, 5-10 µm in diameter) were sealed at both ends to two patch-clamp pipettes. Whole-cell conditions were established for both electrodes with two independent amplifiers (RK400, Bio-Logic, Claix, France) as previously described (Jurevicius & Fischmeister, 1996). However, in the present study, we used both electrodes in the voltage clamp mode to improve voltage clamp homogeneity along the length of the cell. Since the access resistance of the two electrodes was always in the same range (2-5 M $Omega$ ), current applied by both electrodes contributed to a similar extent to the voltage clamp. Thus, the total whole-cell current traces (such as those shown in Fig. 2A) were obtained by adding the current traces provided by the two electrodes. I_Ca was measured by depolarizing voltage pulses (applied to both electrodes) to 0 mV (200 ms) every 8 s from a holding potential of -80 mV. Peak current was determined as the difference between maximal inward current at 0 mV and the current at the end of the 200 ms test pulse. K⁺ currents were blocked by replacing K⁺ with intracellular and extracellular Cs⁺. The fast Na⁺ current was blocked by tetrodotoxin (0.1 mg l^-1). All experiments were performed at room temperature.

Microperfusion of single cardiac myocytes

The double-barrelled microperfusion technique has been described previously (Jurevicius & Fischmeister, 1996). Briefly, after establishing whole-cell recording conditions for both pipettes, the cell was manoeuvred using the two electrodes and positioned in front of the mouth of two adjacent Plexiglas capillaries (square section, 400 µm times 400 µm) separated by a diaphragm with a tip thickness of ~5 µm. Thus, the two halves of the cell could be superfused with different solutions at the same time (S1 and S2 - also used to denote each cell half, as illustrated in the insets in Figs 2, 3, 5 and 6). Mixing of the two solutions was prevented by (i) slightly pressing the cell against the tip of the diaphragm, (ii) positioning the tips of the electrodes inside the mouths of the capillaries, and (iii) maintaining a laminar flow with a velocity of about 1 cm s^-1 by pressure perfusion.

Spatial separation of Ca²⁺ currents of a frog ventricular myocyte

Estimation of the portion of the cell membrane exposed to each solution was performed by perfusing nominally Ca²⁺-free solution (for composition see Solutions and reagents) as S1 or S2 either under control conditions or under application of ISO or FSK. Due to pressure perfusion, solutions could be replaced within 3-5 s, which was demonstrated by an almost immediate decrease in the amplitude of I_Ca upon perfusion with nominally Ca²⁺-free solution. The current reduction upon Ca²⁺ removal in S1 and S2 amounted to total I_Ca. Thus, perfusion of nominally Ca²⁺-free solution as S1 (or S2) allows spatial separation of I_Ca. Since the VDCCs are distributed uniformly in frog cardiomyocytes (Jurevicius & Fischmeister, 1997), I_Ca values measured in S1 or S2 are a direct measure of the membrane under the influence of S1 and S2.

Since the drugs tested here (SNP, SNAP and EHNA) have no effect on the basal I_Ca current (I_basal, i.e. current without ISO or FSK stimulation) (Méry et al. 1993, 1995), drug effects are expressed relative to the stimulatory effect of ISO or FSK (I_I-F) after subtraction of the basal current from the total current (I_total) either in a half-cell or the whole cell:

where I_I-F = I_total,I-F - I_basal and I_drug = I_{total,I-F + drug} - I_basal.

Although ISO-stimulated I_Ca showed a run-down during most experiments, the velocity of the run-down was relatively small (from no run-down to maximal 50 % within 30 min) so that calculation of drug effects could be performed by assuming a linear decline of I_Ca. Thus, drug effects were calculated relative to the interpolated currents from ISO responses prior to the test and the control thereafter. When necessary, currents were expressed as the increase relative to the basal current. Error bars are given as standard errors of the mean (S.E.M.). The number of cells is denoted by n. For statistical evaluation Student's paired or unpaired t test was used, and a difference was considered statistically significant when P < 0.05.

Solutions and reagents

The cardiomyocytes were stored in a solution containing (mM): 99 NaCl, 2.5 KCl, 24 NaHCO₃, 0.5 NaH₂PO₄, 1.8 MgCl₂, 0.9 CaCl₂, 5 creatine, 5 sodium pyruvate and 5 D-glucose, supplemented with 5 µl ml^-1 non-essential amino acids and vitamin solution, 5000 i.u. ml^-1 penicillin and 5 mg ml^-1 streptomycin (pH 7.4, NaOH). The control Ringer solution contained (mM): 107 NaCl, 10 Hepes, 20 CsCl, 4 NaHCO₃, 0.8 NaH₂PO₄, 1.8 MgCl₂, 1.8 CaCl₂, 5 sodium pyruvate, 5 D-glucose and 0.1 mg l^-1 tetrodotoxin (pH 7.4, NaOH). Nominally Ca²⁺-free solution (zero-Ca²⁺ solution) was obtained by replacing 1.8 mM CaCl₂ with 1.8 mM MgCl₂. The patch pipettes were filled with a solution containing (mM): 119.8 CsCl, 5 EGTA (acid form), 4 MgCl₂, 5 creatine phosphate (disodium salt), 3.1 Na₂ATP, 0.42 Na₂GTP, 0.06 CaCl₂, 10 Hepes (pH 7.1, CsOH). All drugs were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France) except: erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA; from Sigma-Aldrich or from Biomol, Hamburg, Germany), tetrodotoxin (from Latoxan, Rosans, France), (±)-S-nitroso-N-acetylpenicillamine (SNAP, from Alexis, Läufelfingen, Switzerland, or Tocris Cookson, Bristol, UK) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO; from Alexis or Tocris Cookson). Forskolin was prepared as stock solutions of 10 mM in 96 % ethanol and each solution tested contained a similar amount of ethanol (< 0.3 %).

RESULTS

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Abstract
Introduction
Methods
Results
Discussion
References

The principal aim of this study was to clarify the spatial organization of NO action upon $beta$ -adrenoceptor-mediated stimulation of I_Ca in frog ventricular myocytes. For this purpose we recorded I_Ca every 8 s applying depolarizing voltage pulses for 200 ms to 0 mV from a holding potential of -80 mV.

The NO donor SNP inhibits ISO-stimulated I_Ca

Figure 1A shows the original traces and time course of I_Ca upon application of the $beta$ -adrenergic agonist ISO (100 nM) to a frog cardiomyocyte voltage clamped by a single electrode. The basal current was increased severalfold by ISO, an effect mediated by an increase in cAMP, resulting in the phosphorylation and upregulation of VDCC by PKA. The additional application of the NO donor sodium nitroprusside (SNP) at increasing concentrations (10 µM, 100 µM and 1 mM) reduced the ISO-mediated stimulation of I_Ca in a dose-dependent and reversible manner until complete block at 1 mM. NO donors are known to increase cGMP levels via activation of the soluble guanylyl cyclase. In frog heart, the increased cGMP antagonizes $beta$ -adrenergic stimulation by activation of PDE2 and subsequent decrease in cAMP levels (Fischmeister & Hartzell, 1987; Méry et al. 1995). Therefore, at each concentration of SNP the selective PDE2 antagonist EHNA (Méry et al. 1995) was added at a concentration of 30 µM (Fig. 1A). As depicted in Fig. 1B, EHNA antagonized the effect of SNP almost completely at 10 and 100 µM, respectively, from 75 ± 6 to 95 ± 4 % (mean ± S.E.M., n = 7) and from 40 ± 7 to 76 ± 7 % (n = 7) of the ISO response, but had only a small effect at 1 mM SNP indicating a shift of the dose-response curve of SNP to larger concentrations (Fig. 1B). Thus, the action of SNP is mediated by PDE2 and not due to possible side effects of cyanide released concomitantly with NO (Friedrich & Butterworth, 1995). A SNP-mediated increase in I_Ca which could be explained by an inhibition of PDE3 upon raising cGMP to low levels (Méry et al. 1993; Kirstein et al. 1995) was not observed.

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Figure 1. The NO donor SNP inhibits the ISO response via activation of PDE2
A, time course of I_Ca in a representative frog ventricular cell voltage clamped using standard single electrode whole-cell recording. I_Ca (filled circles) was recorded by applying a depolarizing pulse to 0 mV for 200 ms every 8 s from a holding potential of -80 mV. The cell was superfused with ISO (100 nM) and subsequently sodium nitroprusside (SNP) at increasing concentrations (10 µM, 100 µM and 1 mM) and EHNA (30 µM) were applied as indicated. Current traces in the upper panel 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 seven experiments as displayed in A. The effects of SNP (open columns) and SNP+EHNA (filled columns) are expressed relative to the increase of I_Ca after pre-stimulation with 100 nM ISO. The columns indicate the means and the error bars the S.E.M. Significant differences determined using Student's paired t test between ISO+SNP and ISO+SNP+EHNA are indicated as: *P < 0.05; **P < 0.01, ***P < 0.001.

Local effect of the NO donor SNP on ISO-stimulated I_Ca

In an earlier study (Jurevicius & Fischmeister, 1996), we introduced a method using double voltage clamp and double-barrelled microperfusion techniques to allow the simultaneous exposure of the two halves of a myocyte to different drugs. Using this method, we have characterized the role of a subcellular cAMP compartment for $beta$ -adrenergic stimulation in frog ventricular myocytes (Jurevicius & Fischmeister, 1996). We have now used this method to describe the effect of local SNP application on an ISO prestimulated cell. The experiment in Fig. 2A (and summary of 5 similar experiments in Fig. 2B) started with an application of ISO (100 nM) to S1, followed by ISO application on the other side (S2) resulting in ISO stimulation of the whole cell (S1+S2). In total, the basal I_Ca of 133 ± 26 pA (density, 1.8 ± 0.4 pA pF^-1, n = 5) was increased by 616 ± 64 % which was a near-maximal response (dose-response curve for ISO not shown). ISO stimulation of S1 alone evoked a response which was slightly larger than 50 % of the total response after stimulation of S1+S2 (Fig. 2B), which is in line with our earlier results (Jurevicius & Fischmeister, 1996). After reaching the maximum response to ISO on S1+S2, 100 µM SNP was applied first to S1 and subsequently to S1+S2 (Fig. 2A). When applied to S1, SNP reduced the total ISO-mediated response by 47 ± 7 % (n = 5), while SNP applied to S1+S2 inhibited the response by 70 ± 10 % (the additional effect at S2 was 23 % of the total ISO response, see also Fig. 2B). Thus, the current decrease caused by SNP is not exclusive to the local side (side of SNP application, S1), but a smaller effect is also observed in the distant side (side without SNP application, S2). Our experiments further demonstrated that this effect did not depend on the order of drug application: indeed, upon removal of SNP on S1 (SNP on S2 remaining), 27 ± 3 % of the ISO response was restored and 43 % after subsequent removal on S2 compared with 70 % of total SNP effect. These experiments clearly show that local application of SNP upon ISO stimulation in frog cardiomyocytes has an effect on both the side of application and the remote part not exposed to SNP. Since the amplitude of I_Ca reflects closely the cAMP concentration (Fischmeister & Hartzell, 1990; Hartzell et al. 1991), our experiments suggest that in cells uniformly exposed to ISO the cAMP concentration is higher on the distant side than on the local side of SNP application. Taken together, local application of SNP induced a cGMP gradient along the cell in the opposite direction to the cAMP gradient. This effect is caused by the SNP-mediated activation of PDE2 (see Fig. 1).

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Figure 2. Half- versus whole-cell exposure to SNP after whole-cell stimulation with ISO
A frog ventricular myocyte was attached at each end to an electrode and positioned so that its approximate mid-point was gently pressed against a thin diaphragm separating channels S1 and S2 of a double-barrelled perfusion system (arrowhead, see schematic drawing). A, the myocyte was first exposed to control extracellular solutions. As shown by the horizontal bars, ISO (100 nM) was first applied to S1, and upon reaching a steady state, ISO was then applied to both sides, S1 and S2, evoking a further increase of I_Ca. Subsequently, SNP (100 µM) was applied to S1 and then to both sides, S1 and S2. Removal of the drugs was performed in reverse order to the sequence of application, showing half-cell application to S2 instead of S1. Current traces in the upper panel 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 five experiments as described in A. The effects are expressed relative to the maximal I_Ca in the presence of ISO on both S1 and S2 (= 100 %). Because of run-down of I_Ca, the effects of SNP were calculated relative to an extrapolated level of the ISO response. Columns indicate the means and the error bars the S.E.M. Significant differences determined using Student's paired t test, denoted by the bracket or relative to ISO stimulation on both sides, are indicated as: *P < 0.05; **P < 0.01, ***P < 0.001.

Spatial effect of a local application of SNP on I_Ca

The approach taken in Fig. 2 did not allow quantification of the spatial contribution of locally applied SNP to both halves of the cell. Therefore, the spatial modulation of ISO-stimulated I_Ca was evaluated by perfusing the local or the distant side with nominally Ca²⁺-free solution.

Figure 3A depicts the time course of a representative experiment. The effect of two different concentrations of SNP (1 and 100 µM) applied to S1 was tested at S1 (local side with respect to SNP application) and at S2 (distant side; note the run-down of I_Ca during the experiment) upon ISO (100 nM) stimulation of I_Ca. At a concentration of 1 µM SNP had no effect on I_Ca, whereas 100 µM SNP showed a pronounced decrease in I_Ca. The spatial effect of SNP was evaluated by short applications of nominally Ca²⁺-free solution at S2 as indicated. The data are summarized in Fig. 3B where local (S1) and distant (S2) I_Ca at the five instances of Ca²⁺ removal are displayed. The dotted line reflects I_Ca in the presence of ISO alone, demonstrating that the run-down is equivalent in S1 and S2. In this experiment, the local I_Ca response to ISO was slightly larger compared with the distant response (55 vs. 45 %); this was probably due to the positioning of the double-barrelled microperfusion system. The representative experiment displayed in Fig. 3 and a summary of several similar experiments (Fig. 4) clearly demonstrate that at all concentrations tested the effect of SNP on I_Ca was predominant at the local (S1) side. The data were normalized with respect to local and distant I_Ca obtained upon application of ISO. In line with our observations in Fig. 1, SNP dose-dependently antagonized the ISO response until almost complete block (to 3 ± 4 % at 1 mM SNP, n = 6) on the local side. In contrast, a much smaller dose-dependent inhibition of I_Ca (to 79 ± 5 % at 1 mM SNP, n = 6) was detected on the distant side (S2). It should be noted that a complete block on the distant side or at least a non-saturating dose-response curve would be expected if SNP or NO diffused directly to the distant side resulting in the activation of soluble guanylyl cyclase in that part of the cell. In contrast, local saturation of guanylyl cyclase and cGMP diffusion along the cell would cause a stable cGMP gradient with a local source (maximum) and a distant sink (minimum). Thus, further increase of local SNP would not affect the cGMP gradient, explaining the apparent saturation of the distant SNP effect. However, to test this hypothesis directly, we examined the effects of carboxy-PTIO, a NO scavenger, on the local and distant responses of I_Ca to a NO donor.

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Figure 3. Local and distant effects of SNP on ISO stimulation
A frog ventricular myocyte was attached at each end to an electrode and positioned so that its approximate mid-point was gently pressed against a thin diaphragm separating channels S1 and S2 of a double-barrelled perfusion system (arrowhead, see schematic drawing). A, the myocyte was first exposed to control extracellular solutions. As shown by the horizontal bars, ISO (100 nM) was first applied on S1 and then on S1+ S2. I_Ca shows a continuous run-down. The total current I_Ca at S1+S2 (filled squares) was separated according to the contribution from both half-cells by omitting Ca²⁺ for S2. Thus, the remaining current is the local I_Ca with respect to SNP (open circles), and the difference between the two current levels is the distant I_Ca (see arrows). The current decreases to 55 % of the total I_Ca, indicating an approximately half-adjustment of the cell between S1 and S2. B, same experiment as in A, showing the effect of two increasing concentrations of SNP (1 and 100 µM) on I_Ca in S1 (local side, open circles) and S2 (distant side, filled circles). Note that the control current in the absence of ISO is subtracted. The dotted lines indicate spontaneous run-down.

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Figure 4. Half-cell exposure to SNP after whole-cell stimulation with ISO: local and distant effects of SNP
Dose-response curves for the local (open circles) and distant (filled circles) effects of SNP on ISO prestimulated cells. Summary of several similar experiments as described in Fig. 3. ISO (100 nM) was applied to the whole cell, whereas SNP was applied in increasing concentrations on a half-cell. The data points show means ± S.E.M. with the number of cells indicated near each symbol. Significant differences between local and distant effects of SNP determined using Student's paired t test are indicated as: ***P < 0.001.

Effect of the NO scavenger carboxy-PTIO on I_Ca at the local and the distant side

Carboxy-PTIO was reported to react rapidly and irreversibly with NO in vitro (Akaike et al. 1993), and was often used as a NO scavenger in vivo in various tissues (for reviews see Yoshida et al. 1994; Maeda et al. 1995). Therefore, carboxy-PTIO should antagonize the inhibitory effect of a NO donor on I_Ca. In the experiment shown in Fig. 5A, I_Ca was increased by application of forskolin (FSK, 1 µM), a direct activator of adenylyl cyclase, to both sides of the cell. We used FSK rather than ISO in this experiment because we found that carboxy-PTIO exerted pronounced anti-adrenergic effects on its own (N. Abi-Gerges, R. Fischmeister & P.-F. Méry, unpublished observations). After I_Ca had reached a steady state, a short application of nominally Ca²⁺-free solution at S2 allowed verification that both sides of the cell contributed equally to total I_Ca. Addition of 1 mM carboxy-PTIO (cPTIO in Fig. 5) applied at S1 had no effect on I_Ca on either side of the cell but prevented the inhibitory effect of a subsequent application of the NO donor SNAP (3 µM) at S1. After washout of carboxy-PTIO from S1, in the continuing presence of SNAP, the total I_Ca current decreased; this was due to a twofold larger inhibitory response of the current at S1 compared with that at S2. However, addition of 1 mM carboxy-PTIO at S2 had no effect on I_Ca on either side of the cell. This indicates that the distant response of I_Ca to SNAP was not due to NO diffusing from the local side where SNAP was applied to the distant side. Figure 5B summarizes the results of a total of seven experiments similar to that shown in Fig. 5A. The data are presented as normalized to the response of I_Ca to 1 µM FSK, which increased total I_Ca by 579 ± 65 %, 48 % of the response deriving from the local (S1) and 52 % from the distant (S2) side. As shown in Fig. 3 and Fig. 4 for SNP on ISO-stimulated I_Ca, local application of SNAP (3 µM) also induced a more pronounced inhibition of FSK-stimulated I_Ca on the local side than on the distant side. Carboxy-PTIO (1 mM) added to the local (S1) side reversed most of the local response to SNAP and attenuated the distant (S2) response so that it was not any more statistically significant. However, addition of the same concentration of carboxy-PTIO to the distant (S2) side modified neither the local (S1) nor the distant (S2) response of I_Ca. These results clearly indicate that the distant response of I_Ca to NO donors was not due to NO diffusing from the local (S1) side where the NO donor was applied to the distant (S2) side but rather to intracellular diffusion of cGMP. Moreover, the distant cGMP level must be smaller than that at the local side, suggesting that the distant half-cell acted as a sink for cGMP.

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Figure 5. Effect of carboxy-PTIO on the local and the distant effect of SNAP after whole-cell stimulation with FSK
A, the myocyte was first exposed to control extracellular solutions. As shown by the horizontal bars, FSK (1 µM) was applied to both cell halves, and SNAP (3 µM) was applied to S1, either in the presence or in the absence of carboxy-PTIO (cPTIO, 1 mM). Thus, S1 became the local side, and S2 became the distant side with respect to SNAP. The total current I_Ca in S1+S2 (filled circles) was separated according to the contribution from both half-cells by omitting Ca²⁺ for S2. Thus, the remaining current is the local I_Ca with respect to SNP (open circles), and the difference between the two current levels is the distant I_Ca (see arrows). The distant effect of cPTIO was also examined by application of the drug to S2. B, summary of seven experiments performed according to the protocol described in A. As shown by the schematic representation at the bottom, FSK was used on the whole cell at 1 µM and the local (S1) and distant (S2) responses of the cell were normalized to 100 %. Subsequently, the local (open bars) and distant (filled bars) effects of FSK were measured under three different conditions: (1) when SNAP (3 µM) was applied to the local (S1) side; (2) when cPTIO (1 mM) was added to SNAP on the local (S1) side; (3) when cPTIO (1 mM) was added to the distant (S2) side. Columns indicate the mean and error bars the S.E.M. of seven experiments. Significant differences determined using Student's paired t test between the conditions tested and the effect of FSK alone are indicated as: *P < 0.05; **P < 0.01.

Estimation of the intracellular cAMP gradient upon local application of SNP

As shown earlier, I_Ca variations can be used as an index of cAMP concentration changes near the VDCCs (Fischmeister & Hartzell, 1990; Hartzell et al. 1991). When exogenous cAMP is perfused inside a frog ventricular myocyte, a relationship is obtained between I_Ca and cAMP concentration which is well fitted by the Michaelis equation (Fischmeister & Hartzell, 1987). Thus, as in our previous study (Jurevicius & Fischmeister, 1996), we assumed that local (E_l) and distant effects (E_d) of ISO on I_Ca reflect the changes in local ([cAMP]_l) and distant ([cAMP]_d) cAMP concentrations, respectively. When normalized to the maximal increase in I_Ca, E_l and E_d were described by the equations: E_l = [cAMP]_l/ ([cAMP]_l + K_D) and E_d = [cAMP]_d/([cAMP]_d + K_D). The dissociation constant (K_D) reflects an 'apparent affinity' of the VDCC for cAMP. However, this parameter disappears when introducing the coefficient $alpha$ = [cAMP]_d/[cAMP]_l, which represents the gradient of distant vs. local cAMP concentration. We thus obtain the equation: $alpha$ = (E_l/E_d)(1 - E_d)/(1 - E_l). In the absence of SNP, with ISO applied on both sides of the cell, $alpha$ = 1. The experimental determination of E_l and E_d from Fig. 4 obtained at 10 µM, 100 µM and 1 mM SNP were used in a reciprocal plot of E_d vs. E_l to derive $alpha$ values of 5, 19 and 122 (Fig. 6), respectively. Thus, local application of SNP creates a strong intracellular cAMP gradient which increases with the concentration of the NO donor. These data provide unequivocal evidence that during local SNP application a large cAMP gradient, similar to the unilateral application of ISO, is observed.

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Figure 6. Estimation of the intracellular cAMP gradient upon local application of SNP
Relationship between the local and the distant effect of SNP. Percentages and error bars are taken from Fig. 4. The dotted line indicates equality of local and distant effects. The values for $alpha$ given near each symbol give an estimate of the relative intracellular cAMP concentration on the distant side ([cAMP]_d) compared with that on the local side ([cAMP]_l): $alpha$ = [cAMP]_d/[cAMP]_l (for further details see text).

Effect of the PDE2 antagonist EHNA on I_Ca at the local and the distant side

Previously published data from our group using 3-isobutyl-1-methylxanthine (IBMX) as a non-selective PDE-antagonist showed that PDEs were located close to the cAMP compartment (Jurevicius & Fischmeister, 1996). The prominent effects of SNP and SNAP on the local side suggested that PDE2 was part of that compartment. Since PDE2 has been reported to hydrolyse not only cAMP but also cGMP (Manganiello et al. 1995), with our next experiments we chose to examine whether PDE2 was involved in the compartmentation of cGMP. Similar to the experiment shown in Fig. 3, 20 nM ISO was applied to the whole cell, and SNP (100 µM) and EHNA (30 µM) were then added to S2 (Fig. 7). On the local side (S1), the effects were identical to the previous experiments and, in addition, EHNA clearly reversed the effect of SNP. Interestingly, the effect of SNP on the distant side (S2) was not reversed by local application of EHNA.

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Figure 7. Effect of half-cell application of EHNA on the distant effect of SNP or SNAP after whole-cell stimulation with ISO
A, the myocyte was first exposed to control extracellular solutions. As shown by the horizontal bars, ISO (20 nM) was applied to both half-cells, and SNP (100 µM) and EHNA (30 µM) were applied to S1. Thus, S1 became the local side, and S2 became the distant side with respect to SNP. The total current I_Ca in S1+S2 (filled squares) was separated according to the contribution from both half-cells by omitting Ca²⁺ for S2. Thus, the remaining current is the local I_Ca with respect to SNP (open circles), and the difference between the two current levels is the distant I_Ca (see arrows). B and C, summary of a number of experiments performed according to the protocol described in A with either SNP (100 µM, n = 3, B) or SNAP (10 µM, n = 3, C). ISO was applied to the whole cell at 10 or 20 nM and EHNA (30 µM) was applied on the same side as SNP (B) or SNAP (C). The currents were separated according to the local (open bars) and distant (filled bars) effects of SNP (B) or SNAP (C). Columns show means and error bars the S.E.M. relative to the response to ISO. The number of individual experiments is given above each column. The left column in each group of three shows the local (left panel) or distant (right panel) effect of SNP (B) or SNAP (C) upon ISO stimulation. The middle column in each group of three shows the effect of EHNA, which reversed the effect of SNP (B) or SNAP (C) on the local side (left panel), but had no effect on the distant side (right panel). The right column in each group of three shows the effect seen upon return to SNP (B) or SNAP (C) on the local (left panel) or distant (right panel) side immediately after removal of EHNA.

The results of a series of three experiments with ISO (20 nM), SNP (100 µM) and EHNA (30 µM) are summarized in Fig. 7B. The same protocol was carried out with 10 nM ISO (n = 3, data not shown) and with SNAP (10 µM) leading to the same results (Fig. 7C). As a control, 30 µM EHNA alone applied after stimulation with ISO caused a much smaller increase, to 119 ± 3 % (10 nM ISO, n = 4) or to 124 ± 10 % (20 nM ISO, n = 4) of the ISO response, indicating a slight basal activity of PDE2. EHNA (30 µM) alone applied on side S1 in the absence of ISO had no effect upon basal I_Ca (n = 5). We conclude that EHNA acted strictly locally and did not diffuse intracellularly to the distant side in amounts large enough to block PDE2. Otherwise, we would have observed at least a partial reversal of the distant effect of SNP. More importantly, the lack of an EHNA effect on the distant side showed that the cGMP gradient caused by local application of SNP or SNAP remained unaffected by EHNA. If the local PDE2 block had increased the local cGMP concentration, we would also have expected an increase in distant (S2) cGMP concentration (due to increased diffusion), and consequently a decrease in distant (S2) I_Ca. Thus, from these experiments we conclude that PDE2 did not hydrolyse the cGMP responsible for the distant effect of NO donors. This supports the idea that PDE2 is located within or in the close vicinity of the cAMP compartment and not distributed uniformly in the cytoplasm.

Localization of PDE2 within the cAMP compartment

The experiments described in Fig. 7 raise the question of the localization of PDE2 relative to the cAMP compartment. IBMX, a broad spectrum PDE inhibitor, largely increased the distant effect of local application of ISO, implying that the cAMP compartment is shielded by PDEs and that cAMP 'escapes' from the cAMP compartment to the cytoplasm upon PDE inhibition (Jurevicius & Fischmeister, 1996). We characterized the SNP effect on the local and distant I_Ca after application of ISO to the cell halves to gain further insight into the action of PDE2. In contrast to the experiments described above, we used cells which were slightly and spontaneously swollen in order to obtain a larger distant effect of ISO. Indeed, we have found recently that the intracellular cAMP compartmentation described in our initial study (Jurevicius & Fischmeister, 1996) is strongly reduced when the cell volume is increased (Jurevicius et al. 1998; J. Jurevicius & R. Fischmeister, unpublished observations). In the typical experiment illustrated in Fig. 8A, ISO (100 nM) was applied to S1 alone (local side) during the whole recording. EHNA (30 µM) was applied to S2 (distant side) in order to block PDE2 on that side. EHNA alone had only a marginal effect on I_Ca upon ISO stimulation. Thereafter, SNP was applied to the local side (S1) at increasing concentrations (10 µM, 100 µM and 1 mM) and exclusively affected the local (S1) response to ISO whereas no effect on I_Ca on the distant (S2) side was observed. Thus, a paradoxical situation was obtained where I_Ca became larger on the distant side of ISO than on the local side, even though cAMP was produced exclusively on the local side. The results of several experiments similar to that described in Fig. 8A are summarized in Fig. 8B. In the first protocol (Prot. no 1), ISO was applied to S1, which increased I_Ca on that side of the cell by 288 ± 68 % (n = 5) above the basal current, while the distant increase in S2 was 58 % of the local increase. During the second protocol (Prot. no 2), SNP (1 mM) was applied to the local side together with ISO. As expected, SNP suppressed the local response of ISO and also the distant effect of ISO. To examine whether this was due to the diminution of cAMP supply from the local to distant side or to activation of PDE2 on the distant side, EHNA was applied on the distant side during the two last protocols. For control (Prot. no 3), EHNA alone on S2 caused a small non-significant increase of the ISO response of 15 ± 7 % (n = 7) on S2, whereas the ISO response on S1 remained unchanged (102 ± 5 %). After local application of 1 mM SNP on S1, with EHNA present on S2 (Prot. no 4), the local ISO response is reduced from 283 ± 45 to 40 ± 9 % (n = 9) above basal I_Ca, whereas the distant effect of ISO is reduced from 204 ± 43 to 114 ± 36 % (n = 9). Thus, the cAMP gradient reversed, because the distant I_Ca became much larger than the local I_Ca. Compared with the results from the protocol without distant EHNA (Prot. no 2), the distant I_Ca increased, showing that the distant PDE2 activation by SNP was responsible for the suppression of the distant effect of ISO. The reversal of the cAMP gradient after local SNP application shows that the cAMP responsible for the distant effect bypasses the local PDE2.

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Figure 8. Effect of SNP on the distant effect of ISO
A, the myocyte was first exposed to control extracellular solutions. As shown by the horizontal bars, ISO (100 nM) was applied to S1 alone, and EHNA (30 µM) was applied to S2 for inhibition of PDE2 on that side. Thereafter, SNP was applied at three increasing concentrations (10, 100 and 1000 µM) to S1. Thus, S1 became the local side, and S2 the distant side with respect to SNP. The total current I_Ca in S1+S2 (filled squares) was separated according to the contribution from both half-cells by omitting Ca²⁺ in S2. Thus, the remaining current is the local I_Ca with respect to SNP (open circles), and the difference between the two current levels is the distant I_Ca (see arrows). Note that, for this type of experiment, a slightly swollen cell was used in order to get a larger distant effect of ISO. B, summary of several experiments as described in A. The local (open bars) and distant (filled bars) effects of ISO (100 nM) are measured in four different protocols: ISO alone on S1 (Prot. no 1); ISO and SNP (1 mM) on S1 (Prot. no 2); ISO on S1 and EHNA (30 µM) on S2 (Prot. no 3); ISO and SNP (1 mM) on S1 and EHNA (30 µM) on S2 (Prot. no 4). Columns show means and error bars the S.E.M. of the number of experiments indicated above each pair of columns. Significant differences determined using Student's paired t test between the local and the distant effect are indicated as: *P < 0.05.

From these experiments, we conclude that there is a tight coupling between the VDCC and PDE2 which 'shields' the Ca²⁺ channels and the PKA. Moreover, the coupling to adenylyl cyclase is weak otherwise cAMP could not bypass PDE2. Thus, PDE2 seems to form a subcompartment around the Ca²⁺ channels responsible for Ca²⁺ channel modulation by cGMP.

DISCUSSION

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Methods
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Discussion
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In the present study, we investigated the modulation of I_Ca by NO donors in frog ventricular myocytes using double voltage clamp and double-barrelled microperfusion techniques for the recording of I_Ca and application of agonists on the local and/or the distant side, respectively. Since the amplitude of I_Ca can be used as a measure of cAMP levels (Hartzell & Fischmeister, 1990; Hartzell et al. 1996; Hove-Madsen et al. 1996), using this approach we could study the effect and the localization of the NO-mediated signalling cascade in relation to cAMP.

In our experiments, the NO donors SNP and SNAP inhibited the ISO- or FSK-mediated stimulation of I_Ca via activation of PDE2 (Méry et al. 1993). The local application of NO donors on ISO-stimulated I_Ca had an effect on both the local and the distant side. On the local side, the NO donors antagonized ISO- or FSK-stimulated I_Ca completely, an effect which could be reversed by the PDE2-selective antagonist EHNA (Méry et al. 1995). Thus, the local effect was found to be identical to the effects described for the whole cell (Méry et al. 1993, 1995) providing no new mechanistic insights. In contrast, on the distant side the I_Ca response to ISO or FSK was inhibited only partially even at saturating local concentrations of SNP (Fig. 4). Thus, local stimulation with NO donors resulted in an almost exclusively homolateral decrease of the cAMP concentration to basal levels, leaving the distant side almost unaffected and resulting in a large gradient of intracellular cAMP along the cell (Fig. 6). Because of the uniformity of ISO stimulation in these experiments, we suggest that the cAMP gradient reflects the inverse of the cGMP gradient.

One could argue that the distant effect of locally applied NO donors is due to NO diffusion rather than to diffusion of intracellular cGMP from one side of the cell to the other. However, in this case, the distant response should become larger as the concentration of the NO donor is increased. Actually, we found that the opposite was true since the distant effect of a local application of SNP saturated at about 20 % inhibition of the ISO response, compared with 100 % inhibition on the local side, when the concentration of the NO donor was increased to 1 mM (Fig. 4). Moreover, the NO scavenger carboxy-PTIO, which was shown in vivo to reverse the effects of NO donors (Akaike et al. 1993; Yoshida et al. 1994; Maeda et al. 1995), was without effect on the distant response to SNAP while it antagonized most of the local response. Therefore, a more likely hypothesis is that the local production of cGMP upon local application of a NO donor determines the amount of cGMP that diffuses to the distant side. Saturation of soluble guanylyl cyclase activity upon activation by large concentrations of NO may set the limit on the amount of cGMP that will diffuse to the rest of the cell.

It has been shown in vitro that PDE2 hydrolyses cAMP and cGMP to the same extent (Beavo & Reifsnyder, 1990; Muller et al. 1992; Lugnier et al. 1992; Manganiello et al. 1995). However, in our experiments, we found that PDE2 did not influence the cGMP distribution along the cell, since local blockage of PDE2 with EHNA did not reverse the distant effects of the NO donors (Fig. 7). Thus, activation or inhibition of PDE2 on one side of the cell does not influence the concentration of cGMP present on the other side. This suggests that there might be two different pools of cGMP. The first is responsible for the local effect of NO donors and might be influenced by the hydrolytic activity of PDE2 (which cannot be observed directly since it is masked by cAMP hydrolysis). The second acts as a source for cytoplasmic diffusion to the distant side and is not influenced by PDE2. In a single pool responsible for both the local and distant effects of NO donors, local blockage of PDE2 should cause an increased distant effect of the NO donors due to increased diffusion of cGMP from the local to the distant side. Both cGMP pools depend on soluble guanylyl cyclase, which might be distributed uniformly. Thus, PDE2 might separate both pools. Because the effects of the NO donors and their reversal by EHNA reflect modulation of cAMP near the VDCC, we conclude that PDE2 must be localized near the cAMP compartment responsible for VDCC phosphorylation rather than distributed over the entire cytoplasm. In this case, cGMP hydrolysis by PDE2 would take place only near the cAMP compartment without or only little affecting the cGMP pool responsible for the distant effect of NO donors. This interpretation is consistent with our data and implies that (i) cGMP production and cGMP action upon PDE2 are spatially separated from each other and (ii) cGMP is less compartmentalized than cAMP.

Using unilateral application of ISO, we found that locally produced cAMP diffuses equally well to the distant half of the cell whether local PDE2 is active or not (compare Prot. no 1 and no 4 in Fig. 6). This indicates that PDE2 is not in contact with the pool of cAMP available for diffusion but only with the compartment of cAMP involved in the regulation of VDCC. From these results, we also conclude that PDE2 shields the VDCC, being more tightly coupled to Ca²⁺ channels than to adenylyl cyclase. Thus, PDE2 may form a subcompartment with PKA and VDCC. Our findings are supported by previous work on the same preparation showing that PDE2 activation masked PDE3 or PDE4 activity (Fischmeister & Hartzell, 1990). Indeed, it was shown that inhibition of PDE3 or PDE4 increased I_Ca after stimulation with ISO or intracellular perfusion with cAMP, but additional PDE2 activation by intracellular cGMP perfusion abolished the effects of PDE3 or PDE4 antagonists (Fischmeister & Hartzell, 1990; Méry et al. 1995). Thus, PDE2 may play a different role from the other two PDE isoforms. Further support for a localization of PDE2 close to the membrane comes from the biochemical work of Lugnier et al. (1992) who showed that in the membrane fraction of frog ventricular myocytes (in contrast to the cytosolic fraction), most of the cAMP hydrolytic activity was due to PDE2 (see also Simmons & Hartzell, 1988).

Our conclusion that PDE2 forms a subcompartment leads to the idea that PDE2 may regulate VDCC phosphorylation separately from other PKA substrate proteins. This may have important physiological implications. In cardiac myocytes, PKA has at least three other main substrates besides the VDCC which control the development and force of contraction. These include phospholamban (Simmerman & Jones, 1998), the sarcoplasmic reticulum (SR) Ca²⁺ release channel (Hain et al. 1995; Valdivia et al. 1995) and troponin I (Rapundalo, 1998). The phosphorylation of phospholamban by PKA increases sequestration of Ca²⁺ into the SR and shortens the systole by dis-inhibition of the sarcoplasmic Ca²⁺-ATPase (Simmerman & Jones, 1998). Troponin I phosphorylation by PKA causing a decrease in the Ca²⁺ sensitivity of the troponin complex, as well as a shortening of the systole (Rapundalo, 1998). PKA phosphorylation of the SR Ca²⁺ release channel may contribute to increase the responsiveness of the channel to Ca²⁺ (Valdivia et al. 1995) and/or to recruit more active channels involved in each contraction cycle of the heart (Hain et al. 1995; see also Marx et al. 2000). In frog ventricular myocytes, there is relatively low content of the Ca²⁺-ATPase-phospholamban complex (Morad & Cleeman, 1987). This is functionally compensated by PKA inhibition of the Na⁺-Ca²⁺ exchanger upon $beta$ -adrenergic stimulation (Fan et al. 1996) suppressing Ca²⁺ influx at later stages of the action potential. Thus, if cGMP decreases VDCC phosphorylation, it would be possible that the cAMP concentration in the compartments relevant for troponin I, SR Ca²⁺ release channel and/or phospholamban phosphorylation (Karczewski et al. 1990) or the Na⁺-Ca²⁺ exchanger (in the frog) remains unaltered. Thus, in the case of $beta$ -adrenergic stimulation, NO would decrease I_Ca but leave other PKA substrate proteins unaffected. Although further experiments are required to test this hypothesis, it may help to reconcile a number of contradictory findings on the regulation of cardiac I_Ca and inotropism by NO and cGMP (for review, see Lohmann et al. 1991; Féron et al. 1999; Shah & MacCarthy, 2000).

REFERENCES

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SHIMONI Y. (1999). Hormonal control of cardiac ion channels and transporters. Progress in Biophysics and Molecular Biology 72, 67-108 [Medline]

SIMMERMAN H. K. B. & JONES, L. R. (1998). Phospholamban: Protein structure, mechanism of action, and role in cardiac function. Physiological Reviews 78, 921-947 [Abstract/Full Text]

SIMMONS M. A. & HARTZELL, H. C. (1988). Role of phosphodiesterase in regulation of calcium current in isolated cardiac myocytes. Molecular Pharmacology 33, 664-671 [Abstract]

STRIESSNIG J. (1999). Pharmacology, structure and function of cardiac L-type Ca²⁺ channels. Cellular Physiology and Biochemistry 9, 242-269 [Medline]

SUMII K. & SPERELAKIS, N. (1995). cGMP-dependent protein kinase regulation of the L-type Ca²⁺ current in rat ventricular myocytes. Circulation Research 77, 803-812 [Abstract/Full Text]

VALDIVIA H. H., KAPLAN, J. H., ELLISDAVIES, G. C. R. & LEDERER, W. J. (1995). Rapid adaptation of cardiac ryanodine receptors: Modulation by Mg²⁺ and phosphorylation. Science 267, 1997-2000 [Medline]

WAHLER G. M. & DOLLINGER, S. J. (1995). Nitric oxide donor SIN-1 inhibits mammalian cardiac calcium current through cGMP-dependent protein kinase. American Journal of Physiology 268, C45-54 [Medline]

WANG Y. G., RECHENMACHER, C. E. & LIPSIUS, S. L. (1998). Nitric oxide signaling mediates stimulation of L-type Ca²⁺ current elicited by withdrawal of acetylcholine in cat atrial myocytes. Journal of General Physiology 111, 113-125 [Medline]

YOSHIDA M., AKAIKE, T., WADA, Y., SATO, K., IKEDA, K., UEDA, S. & MAEDA, H. (1994). Therapeutic effects of imidazolineoxyl n-oxide against endotoxin shock through its direct nitric oxide-scavenging activity. Biochemical and Biophysical Research Communications 202, 923-930 [Medline]

Acknowledgements

We thank Florence Lefebvre for technical help in preparing isolated myocytes, and Patrick Lechêne for computer programming. This study was supported by the PROCOPE program of the European Community.

Corresponding author

R. Fischmeister: INSERM U-446, Universiy of Paris-Sud, Faculty of Pharmacy, 5, Rue Jean-Baptiste Clément, F-92296 Châtenay-Malabry, France.

Email: fisch{at}vjf.inserm.fr

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SIMMERMAN H. K. B. & JONES, L. R. (1998). Phospholamban: Protein structure, mechanism of action, and role in cardiac function. Physiological Reviews 78, 921-947	[Abstract/Full Text]
SIMMONS M. A. & HARTZELL, H. C. (1988). Role of phosphodiesterase in regulation of calcium current in isolated cardiac myocytes. Molecular Pharmacology 33, 664-671	[Abstract]
STRIESSNIG J. (1999). Pharmacology, structure and function of cardiac L-type Ca²⁺ channels. Cellular Physiology and Biochemistry 9, 242-269	[Medline]
SUMII K. & SPERELAKIS, N. (1995). cGMP-dependent protein kinase regulation of the L-type Ca²⁺ current in rat ventricular myocytes. Circulation Research 77, 803-812	[Abstract/Full Text]
VALDIVIA H. H., KAPLAN, J. H., ELLISDAVIES, G. C. R. & LEDERER, W. J. (1995). Rapid adaptation of cardiac ryanodine receptors: Modulation by Mg²⁺ and phosphorylation. Science 267, 1997-2000	[Medline]
WAHLER G. M. & DOLLINGER, S. J. (1995). Nitric oxide donor SIN-1 inhibits mammalian cardiac calcium current through cGMP-dependent protein kinase. American Journal of Physiology 268, C45-54	[Medline]
WANG Y. G., RECHENMACHER, C. E. & LIPSIUS, S. L. (1998). Nitric oxide signaling mediates stimulation of L-type Ca²⁺ current elicited by withdrawal of acetylcholine in cat atrial myocytes. Journal of General Physiology 111, 113-125	[Medline]
YOSHIDA M., AKAIKE, T., WADA, Y., SATO, K., IKEDA, K., UEDA, S. & MAEDA, H. (1994). Therapeutic effects of imidazolineoxyl n-oxide against endotoxin shock through its direct nitric oxide-scavenging activity. Biochemical and Biophysical Research Communications 202, 923-930	[Medline]

				C. Yan, C. L. Miller, and J.-i. Abe Regulation of Phosphodiesterase 3 and Inducible cAMP Early Repressor in the Heart Circ. Res., March 2, 2007; 100(4): 489 - 501. [Abstract] [Full Text] [PDF]

				R. Fischmeister, L. R.V. Castro, A. Abi-Gerges, F. Rochais, J. Jurevicius, J. Leroy, and G. Vandecasteele Compartmentation of Cyclic Nucleotide Signaling in the Heart: The Role of Cyclic Nucleotide Phosphodiesterases Circ. Res., October 13, 2006; 99(8): 816 - 828. [Abstract] [Full Text] [PDF]

				D. A. Heaton, M. Lei, D. Li, S. Golding, T. A. Dawson, R. M. Mohan, and D. J. Paterson Remodeling of the Cardiac Pacemaker L-Type Calcium Current and Its {beta}-Adrenergic Responsiveness in Hypertension After Neuronal NO Synthase Gene Transfer Hypertension, September 1, 2006; 48(3): 443 - 452. [Abstract] [Full Text] [PDF]

				L. R.V. Castro, I. Verde, D. M.F. Cooper, and R. Fischmeister Cyclic Guanosine Monophosphate Compartmentation in Rat Cardiac Myocytes Circulation, May 9, 2006; 113(18): 2221 - 2228. [Abstract] [Full Text] [PDF]