Cicletanine is a dihydrofuropyridine with an original antihypertensive profile, characterized by vascular relaxation, accelerated eicosanoid production, increased natriuresis, and water excretion (1). Recently a protective effect of cicletanine on cardiac reperfusion injury that follows myocardial ischemia was reported (2-4). Burton et al. (5) demonstrated the existence of a significant decrease in ventricular arrhythmias and extent of myocardial necrosis after coronary artery ligation in rabbits treated for 6 weeks with cicletanine. Furthermore, a perfusion of cicletanine (10−5 to 3 × 10−4M) on isolated perfused heart was shown to reduce the intracellular Na+ (Nai+) and Ca2+ (Cai2+) overload observed after aortic cross clamping and subsequent reperfusion, suggesting a modulation by cicletanine of the sarcolemmal ion-transport system (6). It is well known that the cardiomyocyte Na+-H+ exchanger may play an important role in the Nai+ and Cai2+ overload associated with the reperfusion process, and that its inhibition may result in protection against the reperfusion cell-injury process (7). The aim of this study was to determine the effect of cicletanine on the regulation of pHi through the Na+-H+ exchanger system in a primary culture of chick embryonic cardiomyocytes. Part of this study was published in abstract form (8).
Ventricular myocyte culture
Layers of culture of ventricular myocytes were prepared from 11-day-old chick embryos by using the technique previously described (9). Ventricles were minced, placed in Ca2+-Mg2+-free phosphated buffered solution (PBS) with 0.05% trypsin (wt/vol) at 37°C and agitated for 7 min. The supernatant was resuspended in medium 199 (M199) with Hanks' solution (Eurobio, Paris, France), added with 20% heat-inactivated newborn calf serum (NCS). The cycle was repeated 7 times, and the suspension was centrifuged at 2,500 rev/min for 15 min at 4°C. The cells were resuspended in M199 with 5% heat-inactivated NCS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 2 mM glutamine, and 25 mM NaHCO3. The suspension was filtered, and fibroblasts were eliminated by differential attachment (9). Cells were cultured at a final density of 3 × 105 cells/ml on glass coverslips, in a humid 5% CO2/95% air atmosphere at 37°C.
Measurement of pHi
The pHi was assessed in single cells by using fluorescence imagery (10). Cells were loaded with BCECF (Molecular Probe Inc., Eugene, OR, U.S.A.), by incubation for 45 min at 37°C with the acetoxymethyl ester form of the dye (7.5 μM) in M199. The cells were then washed twice, and the coverslip was placed in a cell chamber, mounted on the stage of an inverted microscope (Nikon Diaphot, Tokyo, Japan) fitted with a cooled integrating CCD imaging system (Newcastle Photometric System, Newcastle, U.K.). The cells were continuously superfused at a flow rate of 1 ml/min, with Na+-HEPES solution containing (in mM) 143.0 NaCl, 4.6 KCl, 0.8 MgSO4, 0.8 KH2PO4, 1.0 CaCl2, 5.6 glucose, 5.4 HEPES, and 4.6 TRIS (pH 7.4 at 37°C). The cells were allowed to equilibrate for 10 min before the start of the experiment. Cells were illuminated alternately at 440 and 490 nm, and the intensity of emitted light at wavelengths >520 nm was collected. Recordings were made from single cells over a 500-ms period at each excitation wavelength. The CCD imaging system allowed simultaneous measurements to be made from ≤16 cells in a field of view. The ratio of the emitted fluorescence at 490 nm/440 nm (F490/440) was calculated as a function of time for each cell.
Calibration of pHi
The pHi was estimated with a calibration curve, established for each individual cell by using the nigericin technique (11). In brief, at the end of each experiment, cells were exposed to a calibration solution containing (in mM) 110 KCl, 1.0 MgCl2, 1.0 CaCl2, 20 HEPES, 30 choline chloride, and 10 nigericin. In the presence of ionophores, internal [H+] would tend to equilibrate with external [H+]. By using solutions in which pH was adjusted to different values ranging from 6.3 to 8.0, the linear function between F490/440 and external pH could be determined.
Assessment of pHi regulatory mechanisms
The pHi regulatory mechanisms were studied during pHi recovery from an acute intracellular acid load, induced by transient exposure to NH4Cl (10 mM; 12). The pHi recovery was assessed in the presence and in the absence of external Na+ (Na+-free solution, where NaCl is replaced by N-methylglucamine) and after cell incubation with N,N-hexamethyle-namiloride (HMA; 10−5M, 30 min) or with 4,4′-diisothio-cyanatostilbone-2,2′-disulfonic acid (DIDS; 10−4M, 30 min). Calculation of initial pHi recovery rate was done by using linear-regression analysis by Newcastle Photometric System soft-ware. Cell-buffering capacity (βi) was determined for each individual cell from the variations in pHi consequent to NH4Cl addition and removal, as described previously (12).
Effect of cicletanine on pHi control
Cicletanine, at concentrations ranging from 10−7 to 10−5M, was added to cells just after BCECF-AM loading and allowed to equilibrate for 60 min before experiment.
Results are presented as mean ± SD. Statistical analysis was performed by using the nonparametric Kruskal-Wallis test, with Statview 4.1 software (Abacus Concepts, Inc., Berkeley, CA, U.S.A.). A value of p ≤ 0.05 was considered significant.
pHi Recovery from an acid load in control cells
A consistent pattern of pHi recovery after acute acid load was observed in control cells (n = 67). Initial rate of pHi recovery in Na+-HEPES solution was 0.033 ± 0.011 pH unit/min, at a basal pHi of 6.96 ± 0.08 pH units. Conversely, in Na+-free medium, no pHi recovery could be observed, as shown on a typical experiment in Fig. 1. Incubation of cells with HMA (10−5M) induced a significant inhibition of pHi recovery as compared with control cells (0.004 ± 0.002 pH unit/min, n = 8, p < 0.01 vs. control; Fig. 2). In contrast, the stilbene derivative DIDS (10−4M) did not significantly modify the Na+-dependent pHi recovery from an acid load as compared with control cells (0.033 ± 0.010 pH unit/min, n = 11; p, not significant).
Effect of cicletanine on pHi regulation
No significant difference in basal pHi could be observed after cell incubation in the presence of cicletanine at concentrations ranging between 10−7 and 10−5M. Cicletanine at 10−7M induced a significant decrease in the Na+-dependent pHi recovery from an acid load induced by transient exposure to NH4Cl, whereas at higher concentrations ranging from 10−6 to 10−5M, a significant stimulation of this recovery mechanism was observed (Fig. 2). In cells exposed to 10−5M cicletanine, addition of HMA (10−5M) significantly reduced the Na+-dependent pHi recovery from acid load as compared with cells not exposed to HMA (0.003 ± 0.002 pH units/min vs. 0.090 ± 0.030 pH units/min, n = 16, p < 0.001), as shown in Fig. 2. In contrast, in cells exposed to 10−5M cicletanine, addition of DIDS (10−4M) was without a significant effect on pHi recovery as compared with cells not exposed to DIDS (0.085 ± 0.020 pHi units/min vs. 0.090 ± 0.030 pHi units/min, n = 18; p, not significant). The intracellular buffering capacity (βi), determined in individual cells at the same mean pHi of 6.96 pHi units, was not significantly modified in the presence of different concentrations of cicletanine as compared with control cells (βi in control cells, 10.1 ± 1.2 mM vs. βi in cells exposed to cicletanine 10−5M: 8.95 ± 2.7 mM; p, not significant).
Our working hypothesis was that an inhibition of the Na+-H+ exchanger could be implicated in the protective effect of cicletanine on the consequences of reperfusion after myocardial ischemia. This hypothesis relies on previous studies showing that cicletanine (10−5 to 3 × 10−4M) reduces the increase in Na+ and Ca2+ cell content observed after aortic cross-clamping and subsequent reperfusion on isolated perfused heart (3,6). The Na+-H+ exchanger could be implicated in this latter phenomenon, as it participates in the regulation of Cai2+ via the regulation of Nai+ and the Na+-Ca2+ exchanger (7,13). This link between Cai2+ and the Na+-H+ exchanger was reported to be related to cell injury after reperfusion of ischemic myocardium (14,15). Thus inhibition of the exchanger with amiloride derivatives was shown experimentally to preserve cellular integrity in reperfused heart tissue (13).
The main result of this study was that cicletanine exerted a dual effect on pH recovery from acid load in cultured chick embryonic cardiomyocytes. At low concentration (10−7M), cicletanine significantly depressed this recovery, whereas at higher concentrations (10−6-10−5M), cicletanine significantly stimulated this mechanism. In our experimental conditions, this recovery is Na dependent and mainly mediated by the Na+-H+ exchanger, as it is inhibited by amiloride derivative HMA (10−5M). These results strongly suggest that cicletanine exerts a dual effect on the Na+-H+ exchanger and could reflect the complex pleiotropic biochemical regulation of the exchanger reported in several cell lines. For example, in proximal tubular cells of the kidney, angiotensin II at low concentrations (10−12-10−10M) was shown to stimulate the Na+-H+ exchanger, whereas higher concentration of angiotensin II (10−7M) resulted in an inhibition (17). This biphasic modulation may reflect the multiple signaling pathways regulating the Na+-H+ exchanger (18). Thus several studies have shown that the activity of the Na+-H+ exchanger was stimulated by Cai2+, protein kinase C, and cyclic guanosine monophosphate (cGMP). Actually, cicletanine was reported to decrease the Cai2+, to inhibit the protein kinase C and the cGMP phosphodiesterase, thus increasing the cellular cGMP content (19). This latter modulation of cyclic nucleotides could be responsible for the dual effect observed with cicletanine, leading to either an inhibition or a stimulation of the Na+-H+ antiport activity, depending on the concentration used.
One other possibility could be that cicletanine could affect the Na+-HCO3− cotransport. This mechanism is known to couple Na+ and HCO3− in relation to their respective gradients and is sensitive to stilbene derivatives such as DIDS or SITS (16,20). Although our solutions are nominally HCO3 free, endogenous formation of CO2 could contribute to the activation of this transporter. In this study, the effects observed with cicletanine are DIDS insensitive, thus suggesting that Na+-HCO3− cotransport is not implicated in pH recovery. However, the participation of a DIDS-insensitive form cannot be excluded. In relation to this, a DIDS-insensitive Cl−-HCO3− exchanger was reported in cortical collecting duct cells (21). On the other hand, Fanous et al. (22) reported that cicletanine inhibits a Na+-HCO3 cotransporter in A10 cells. Thus if such a DIDS-insensitive cotransporter is present in heart myocytes, it could contribute to pHi recovery from acid load, and its inhibition by cicletanine could account for the results observed at low doses of this compound. Further experiments are required to test this latter hypothesis.
The result of our study failed to show a decrease in Na+-H+ exchanger activity at concentrations of cicletanine close to therapeutic concentrations (10−6 to 8 × 10−6M), and suggested that a direct inhibition of the Na+-H+ exchanger by cicletanine might not be involved in the protective effect of this agent from ischemia/reperfusion injury (23). Other mechanisms could account for this effect. In this regard, Jouve et al. (2) suggested that cicletanine could protect the hearts of anesthetized dogs against arrhythmias in correlation with an increased prostacyclin (PGI2) generation. Ferdinandy et al. (4) recently proposed that the opening of ATP-dependent K+ channels may be responsible for the antiarrhythmic effects of cicletanine. However, none of these mechanisms can account solely for the significant myocardial protection of cicletanine, and further studies are required to elucidate this problem. On the other hand, cicletanine did not alter the cellular buffering capacity at all concentrations tested.
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