Nicorandil is a vasodilating agent with two postulated mechanisms of action: an increased potassium conductance of the myocytes (1) by activation of adenosine triphosphate (ATP)-sensitive K channels (2) and a nitrate-like mode of action with an increased intracellular cyclic guanosine monophosphate (cGMP) induced by activation of the guanylyl cyclase (3). These mechanisms have been shown to act differently on different parts of the vascular system (4-6), the nitrate-like effect of nicorandil inducing the vasodilation of large coronary arteries, and its opening action on K-ATP channels dilating the coronary resistance vessels.
The coronary vasodilating actions of nicorandil prompted its use in the treatment of coronary artery disease (7) because its negative inotropic effects are small (8) and probably much smaller than those of calcium channel antagonists such as diltiazem or nifedipine. Indeed, in a recent study of the myocardial inotropic effect of nicorandil in human tissue, Müller-Ehmsen et al. (9) showed a significant negative inotropic action of the drug, but this effect appeared only for high concentrations, much larger than those used in patients and much larger than those of diltiazem or nifedipine that produce the same negative inotropic effect.
Because the increase in K+ extrusion outside the cell induced by the increased K+ conductance decreases the duration of the action potential and decreases Ca2+ entry inside the cell, a possible mechanism of the small but significant negative inotropic effect of nicorandil may be a decrease in the calcium transient. The goal of our study was thus to evaluate on rat cardiomyocytes the effect of nicorandil on cytoplasmic calcium transient by using indo-1/AM. To evaluate whether a change in calcium transient induced by the drug could be attributed to its NO-donor properties or to the opening of K-ATP channels, we also evaluated its effects when given with glibenclamide, a potassium channel antagonist, and we evaluated the effects of a pure K-ATP channel opener, aprikalim, and those of a NO donor, 3-morpholino-sydnonimine (Sin-1).
Cardiac myocytes were obtained from the hearts of male Wistar rats (270-300 g). Cardiomyocytes were isolated by using the procedure of Powell and Twist (10), with slight modifications. After anesthesia of the rat, a thoracotomy was performed. The heart was excised, the aorta was cannulated, and the heart was perfused under a Langendorff column, allowing a retrograde perfusion of the heart for 5 min by a calcium-free solution containing (mM) NaCl, 35; KCl, 4.8; KH2PO4, 1.2; Na2HPO4, 16; NaHCO3, 25; sucrose, 134; glucose, 10; HEPES, 10. This was followed by the infusion of the same buffer in which collagenase A (1.2 mg/ml) was added.
After 40 min of perfusion, the heart was taken from the column and cut into small (1 mm) pieces in the same calcium-free buffer. Cells were filtered, washed, and calcium (1.2 μM) was reintroduced. At the end of this procedure, ∼80% of myocytes had a normal architecture, were quiescent, and could be electrically stimulated. Cardiomyocytes were then resuspended in a culture medium (BM86 Wisler) and placed on Petri dishes (31-mm diameter), which had been specially prepared for the imaging procedure with a hole (17-mm diameter) cut at the bottom of the dish and a glass coverslip (22 × 22 mm, 0.13-0.16 mm thick) glued on the bottom of the dish. The volume of liquid in the dish was 2 ml. This volume plus the volume of liquid included in the perfusion tube (1 ml) was flushed before each drug injection. Cells were incubated on these dishes previously coated with laminin (30 μM/ml) for 3 h at 37°C in an incubator with room air supplemented with 5% CO2. At the end of this period, cells were adherent on the bottom of the dish, and the culture medium was changed. The cells were used for the study either at that time or 12 h later.
Loading with indo-1/AM
Before each experiment, the cells were preloaded for 15 min at room temperature with indo 1/AM by incubation in 200-μl culture medium containing 10 μg indo-1/AM, 180 μl bovine serum albumin, and 5 μg pluronic acid in 5 μl dimethylsulfoxide (DMSO). The cells were then washed and maintained at room temperature for 40 min in the culture medium before the fluorescence measurements. The nutrient medium was replaced with a Krebs solution containing (mM): NaCl, 117; KCl, 5.7; NaHCO3, 4.4; KH2PO4, 1.2; MgCl2, 1.7; CaCl2, 1.25; HEPES, 21; glucose, 10; creatine, 10; taurine, 20; and buffered to pH 7.4.
Cytosolic calcium measurements were carried out by dual-emission microfluorometry with the indo-1/AM probe (Hamamatsu, Massy, France). The cell, loaded with the fluorescent probe, were excited through a ×40 oil-immersion objective by using a 100 W xenon light, neutrally attenuated to avoid bleaching, and filtered at 360 nm. Excitation and emission beams were separated by a 380-nm dichroic filter. Emission spectra were then divided into two halves by a 455-nm dichroic filter. From the two halves of the indo-1 emission spectra, two signals were selected by interference filters at 405 and 480 nm. These signals were recorded by photometers and passed to an amplifier. The fluorescence ration F405/F480, which is independent of the probe concentration, was directly calculated from both signals. All optics and photometers for indo-1 were obtained from Nikon-France (Paris, France).
Compound application and electrical stimulation
A special apparatus was built to allow the perfusion of drugs and electrical stimulation of the cells. This apparatus (30 mm diameter), to be placed on the top of the Petri dish, included two semicircular iridium-platinum electrodes and two tubes that could be connected to a perfusion apparatus. Electrical pacing was performed by using a Harvard stimulator delivering 40-V stimuli of a duration of 10 ms at a frequency of 1 Hz. Cells were continuously perfused at a rate of 2 ml/min by the Krebs solution at room temperature. The solution had been previously bubbled by a gas mixture (95% O2, 5% CO2). We previously verified that when this procedure is used, pH is not modified for ≥2 h.
Each cell isolation allowed the seeding of ∼30 Petri dishes. One to three cells could be observed in each field of the microscope. A continuous pacing was then performed. Data were recorded for 8.52 s (72 image acquisitions separated by 120-ms intervals). Although the sampling rate was small, we chose this procedure to have a continuous recording instead of a single beat because the number of images is, in this system, limited to 72 per acquisition. We previously verified that this procedure did not produce a significant attenuation of the measured peak of the calcium transient by comparing it with that obtained at the maximal rate of acquisition of the system (50 Hz, that is, 72 points separated by 20-ms intervals). After recording of the control state, the bath inside the Petri dish was aspirated and replaced by the solution of the drug to be tested, which was continuously infused for 10 min under continuous pacing. A recording was then performed. Each Petri dish was used for one concentration of a drug. Drugs (nicorandil or aprikalim alone or with glibenclamide 10−5M, or Sin-1) were infused in random order at drug concentrations of 10−7, 10−6, 10−5, and 10−4M. A 10-min continuous pacing under the perfusion of Krebs solution was performed after each series of drug concentrations to verify the effect of pacing alone on cytosolic calcium concentration.
The mean ratio image of each cell was calculated by the average of all points included in the area of the cell (Fig. 1). The transformation of 405/480 ratio values into calcium concentrations takes into account Kd values and concentrations of calcium but also those of other ions, particularly H+. Because ion concentrations are different in different parts of a cell and in different conditions, the use of a fixed formula to obtain absolute values of calcium concentrations may be misleading. Thus rather than trying to express cellular calcium concentration in absolute values, we preferred to present only the 405/480 ratio values as indices of calcium concentrations. Two time points were measured: end diastole (at the bottom of the calcium increase) and the peak of the calcium ratio (systolic calcium). The calcium transient (or Δ ratio) was the difference between these two points. For each recorded period of the experiments (control or intervention), considered values were the mean of the regularly paced beats.
Data are presented as mean ± SEM. For comparing values obtained during control and during an intervention, statistical analysis was performed by using a paired t test because only one intervention was produced after a control.
The absolute values of diastolic calcium (expressed as the 405/480 ratio) and those of the calcium transient (systolic-diastolic 405/480 ratios) are given in Table 1.
Effect of continuous ventricular pacing
Continuous ventricular pacing for 10 min induced a significant increase in diastolic calcium (Table 1). The calcium transient tended to decrease slightly, but this change was not statistically significant (Table 1).
Effect of aprikalim
For doses of aprikalim from 10−7 to 10−4M, the calcium transient was not significantly modified by any dose (Table 1; Fig. 2). The difference of cell behavior between pacing alone and pacing with aprikalim is displayed in Fig. 2, which shows an absence of significant diastolic calcium increase under continuous pacing except for the 10−6M dose of the drug, but the difference with pacing alone was not statistically significant.
Effect of nicorandil
Similar to the results obtained with aprikalim, with nicorandil diastolic calcium did not increase significantly during continuous pacing, except for the largest dose, and for the 10−6M dose and the calcium transient was not significantly altered (Table 1; Fig. 3).
Effect of nicorandil and aprikalim in association with glibenclamide
To discriminate the mechanisms of action of nicorandil and aprikalim due to K-ATP channel-opening properties of the drug from those due to its NO-donor properties, the same experiments were performed with the addition of the K-ATP channel blocker, glibenclamide (10−5 M3). In a first series of experiments, it was found that glibenclamide alone produced minor changes in diastolic ratio and in delta ratio, which were not different from those induced by pacing alone (Table 1). In a separate series of four experiments, it also was verified that the association of glibenclamide (10−5M) with doses of aprikalim (10−7 to 10−4M) did not significantly modify diastolic ratio and delta ratio. For increasing doses of aprikalim (10−7 to 10−4M) in association with glibenclamide, diastolic ratio changes were +0.19, +0.30, +0.018, and +0.16, respectively, and the changes in delta ratios were +0.03, −0.15, −0.01, and −0.18, respectively. These changes were not significantly different from control and from those induced by pacing alone (Table 1). In contrast, with nicorandil plus glibenclamide, there was a decrease (by ∼20%) of the calcium transient for all studied doses (Table 1, Fig. 4).
Effect of Sin-1
The effects of NO on free cellular calcium were analyzed by the infusion of the NO-donor Sin-1. The effects were similar to those of nicorandil given in association with glibenclamide, with an increase in diastolic calcium to the same extent as that observed with pacing alone, and there was a significant decrease in calcium transient (by ∼20%) for all studied doses (Table 1; Fig. 5).
The principal results of this study can be summarized as follows:
- K-ATP channel openers aprikalim and nicorandil tend to prevent the increase in diastolic calcium induced by a regular pacing in isolated cardiomyocytes without decreasing the calcium transient.
- NO induces a modest but significant decrease in calcium transient.
- The NO-donor properties of nicorandil are not apparent when it is given alone but are unmasked when the K-ATP channel-opening properties of the drug are blocked.
The use of nicorandil was proposed for the treatment of coronary artery disease because of its vasorelaxant properties associated with a protection against coronary spasm (11) and decreased ischemia/reperfusion injury of the heart in reversible and irreversible experimental coronary artery occlusion (12). In addition, its negative inotropic properties were shown to be smaller than those of other treatments of coronary artery disease, such as Ca2+ channel blockers, with similar vasorelaxant properties (9). Clinical studies were thus undertaken to compare nicorandil with calcium channel blockers in patients with coronary artery disease (13,14).
The decreased ischemia/reperfusion injury induced by nicorandil was attributed to a decreased intracellular Ca2+ concentration, protecting the myocyte against the ischemia-induced calcium overload (12). This was never investigated previously, but it was shown in canine coronary artery smooth muscle that nicorandil and the pure potassium channel opener cromakalim did decrease the intracellular Ca2+ concentration measured by fura-2 as calcium indicator (8). This effect was attributed to the potassium channel-opening properties of the drug because potassium extrusion outside the cell induced by these drugs produces a cellular hyperpolarization, which closes the Ca2+ channel and decreases the intracellular Ca2+ concentration. In this study, no decrease in diastolic calcium was found with either nicorandil or the pure potassium channel opener aprikalim (Table 1; Figs. 2 and 3). However, continuous pacing of the cells without any drug induced a significant increase in diastolic calcium (Table 1), suggesting some metabolic imbalance of these cells in these in vitro conditions. Our data show that there was a tendency toward a protection by these drugs against the calcium overload induced by continuous cellular pacing (Table 1; Figs. 2 and 3). Nicorandil and aprikalim appear thus as protecting, at least partially, the cells against calcium overload, but because the difference with pacing alone was not statistically significant, our results suggest that nicorandil and aprikalim may protect, at least partially, the cells against calcium overload. However, because no clear dose-response relation was observed, it is not possible to conclude from our data which concentration may be pharmacologically relevant.
Although the negative inotropic properties of nicorandil have been shown to be small (9), a decrease in calcium entry inside the cell may produce a decrease in calcium. Our results show that this was not the case because both nicorandil and aprikalim did not induce any significant decrease in calcium transient (Figs. 2 and 3).
Besides its potassium channel-opening properties, nicorandil also acts as a NO donor (1), increasing cGMP concentration in the cell (15,16). The possible negative effect of cGMP on cardiomyocytes is still a matter of debate because some studies showed a clear negative effect (17-19), whereas others (20-22) did not demonstrate any significant contractile effect of increased cGMP levels. In the study of contractile changes induced by nicorandil on human cardiac tissue, Müller-Ehmsen et al. (9) attributed the small negative inotropic effect of the drug to its potassium channel-opening properties only with no negative inotropic effect of increased cGMP levels. In our study, we found that the pure NO donor Sin-1 induced a significant decrease in calcium transient by ∼20% (Table 1; Fig. 5). This result is similar to that found by Werich et al. (22). When the potassium channel-opening properties of nicorandil were blocked by the potassium channel-antagonist glibenclamide (Table 1; Fig. 4), the same decrease in calcium transient as that found with Sin-1 was obtained, and the protection against the increase in diastolic calcium induced by nicorandil disappeared, masking thus the potassium channel-opening properties. Conversely, its NO-donor effects were not apparent when the potassium channel properties were present. It can be assumed that, when both effects are present, the prevention of calcium overload induced by potassium channel opening could improve calcium homeostasis of the cell and could counterbalance the negative effect of increased cGMP production induced by the NO-donor effects of nicorandil.
Acknowledgment: We gratefully acknowledge Laboratoire Bellon, France, for generously providing us with nicorandil and aprikalim.
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