Regulation of intracellular pH in cardiac cells relies on the activity of various sarcolemmal transport mechanisms, i.e., the Na+/H+ exchange, an Na+, HCO3--dependent mechanism, and the Na+-independent Cl-/HCO3- exchange. Although Na+/H+ exchange and the Na+, HCO3--dependent transport are both activated by an intracellular acidification (1,2), then responsible for acid(-equivalent) efflux, Cl-/HCO3- exchange acts as an acidifying mechanism by extruding HCO3- ions (3). Steady-state pHi is therefore the result of a balance between the activity of these pHi-regulating mechanisms and the background acid loading of the cell (4). In this context, the specific inhibition of one of these mechanisms would lead to a shift in the steady-state pHi toward either alkaline or acid values. Such a shift might be beneficial in some pathological conditions, such as ischemia. Indeed, we may suppose that if steady-state pHi were, for instance, more alkaline (after Cl-/HCO3- exchange inhibition), the metabolic H+ production related to ischemia would draw pHi toward a less acid level than that reached when preischemic pHi is “normal” and could then afford some protection against the development of ischemic alterations by decreasing the availability of protons for subsequent activation of the dual acid extrusion system. Moreover, as previously demonstrated for Na+/H+ exchange (5), the Na+, HCO3--dependent transport might play a role in the reperfusion-related alterations since its activation would, as for Na+/H+ exchange, lead to Na+ entry and consequently to Ca2+ overloading of the cardiac cells in such conditions. Therefore, inhibiting this transport specifically could further help the heart to recover during reperfusion after an ischemic episode.
Whereas several specific inhibitors of Na+/H+ exchange are now available (6-8), no specific inhibitor of either Cl-/HCO3- exchange or the Na+, HCO3--dependent transport is yet available. To detect one such inhibitor, we tested the effects of S20787 on the pHi-regulating mechanisms in rat isolated ventricular myocytes. We recorded intracellular pH with the fluorescent probe carboxy-SNARF-1.
MATERIALS AND METHODS
Male Wistar rats weighing 250-300 g were used. All procedures were in accordance with the regulations of the Ministère de l'Agriculture et de la Forêt, France, for the care and use of laboratory animals.
Isolation of rat ventricular myocytes
Single ventricular myocytes were obtained from the hearts of rats (anesthetized with pentothal 50 mg/kg body weight intraperitoneally, i.p.) with a combination of enzymatic (collagenase 0.28 mg/ml, Yakult, Japan; protease type XIV 0.05 mg/ml, Sigma Chemical, St Louis, MO, U.S.A.) and mechanical dispersion. The composition of the basic solution and further details of the procedure were described previously (9). Calcium-tolerant, rod-shaped ventricular myocytes were used on the day of isolation.
HEPES-buffered Tyrode's solution contained (in mM) NaCl 140, KCl 5.4, CaCl2 1, MgCl2 1.2, glucose 11, and HEPES 10, pH adjusted to 7.4 at 37°C with NaOH. In Na+-free HEPES-buffered medium, NaCl was replaced with 140 mM N-methyl-D-glucamine (NMG) and the pH was adjusted to 7.4 with HCl. In bicarbonate-buffered Tyrode's solution, the sodium chloride concentration was reduced to 117 mM and 23 mM NaHCO3 was substituted for HEPES. This solution was equilibrated with 5%CO2/95%O2 and had a pH of 7.4 at 37°C. Ammonium chloride or sodium acetate (both Sigma) was added directly to solutions with osmotic compensation. Dimethyl amiloride (DMA 50 μM; Sigma), an inhibitor of Na+/H+ exchange, was first dissolved in dimethyl sulfoxide (DMSO) before being added to Tyrode's solution (DMSO concentration <0.08%). 4,4′-Diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS 200 μM, Sigma) was added, as solid, to solutions soon before their use. Nigericin calibration solutions used in this study were described previously (2).
Drug application protocol
S20787 was obtained from the Institut de Recherches Internationales Servier, France. This drug was first dissolved in DMSO before being added to Tyrode's solution, and its final concentration ranged from 10-11 to 5.10-6M (molecular weight = 367). The maximum DMSO concentration used in the present study was 0.1%. We could not test >5.10-6M concentrations of S20787 because to achieve dissolution of the drug in that case we had to use higher DMSO concentrations, which then significantly affected pHi regulation. S20787 was applied to myocytes for 15-20 min before the pHi recovery was obtained after either an acid or an alkaline load and was present throughout the period of pHi recording.
Measurement of pHi
The pHi of single isolated myocytes was monitored with the pH-sensitive fluorescent dye carboxy-SNARF-1 (carboxyseminaphtorhodafluor; Molecular Probes, Eugene, OR, U.S.A.) (10). Cells were loaded with SNARF by incubating them in a 5-μM solution of the acetoxy-methyl ester for 10 min at room temperature.
Carboxy-SNARF-1 flourescence from individual cells was measured with an inverted microscope (Nikon Diaphot) converted to epifluorescence. SNARF-loaded cells were excited with light at 515 nm, and the resulting fluorescence at 590 and 640 nm was measured with two photomultiplier tubes (Nikon, France). The signals were then digitized at 0.5 kHz (Cambridge Electronic Design, CED 1401 intelligent interface, Cambridge, U.K.) and stored for later analysis on the hard disk of a computer. The emission ratio 590/640 obtained from intracellular SNARF was calculated and converted to a linear pH scale by in situ calibration data obtained at the end of the experiment by the nigericin technique, as described previously (10,11). Finally, the calibrated pHi signal was averaged over 0.5-s intervals.
Estimation of intracellular intrinsic buffering power at different pHi
The method used to estimate intracellular intrinsic buffering power (βi) was described previously (2,12). A stepwise reduction of external NH4Cl (from 20 mM) was applied to a selected myocyte. Each reduction in NH4+ resulted in the generation of intracellular H+, due to dissociation of NH4+ into H+ ions and NH3, with subsequent efflux of NH3(13). The resultant changes in pHi were used to estimate βi as follows: βi = Δ[NH4+]i/ΔpHi, where [NH4+]i = ([H+]i × [NH4+]o)/[H+]o. The experiments were performed without extracellular Na+ to prevent acid extrusion and barium (1 mM) to reduce NH4+ efflux through potassium channels (12).
Calculation of sarcolemmal acid flux
Details of the method for calculating acid flux (JH) during pHi recovery in ventricular myocytes were described previously (2). Acid flux was estimated with the following equation: JH = βT · dpHi/dt, where βT is the total intracellular buffering power and dpHi/dt is the rate of pHi recovery at any given pHi. In HEPES-buffered medium, βT equals the intrinsic buffering power βi. In the present study, βi at any given pHi was calculated with the following equation (empirical description of the dependence of βi on pHi under control conditions): βi = 127.5-13.3 × pHi (data fitted by linear regression analysis). In HCO3--buffered Tyrode's solution, the term βT comprises the sum of βi plus βCO2, the buffering power caused by intracellular CO2/HCO3-, with βCO2 = 2.3 [HCO3-]i (this assumes that the myocyte behaves as an open system for CO2).
All data are mean ± SEM along with the number of observations (n). Student's t test or one-way analysis of variance followed by Newman-Keuls test was used to test the effects of S20787. Differences were considered significant at the level of p < 0.05.
Effect of S20787 on intracellular intrinsic buffering power (βi)
Figure 1A shows a typical experiment performed to estimate the pHi dependence of βi in a single rat isolated ventricular myocyte. pHi was recorded using carboxy-SNARF-1. The different jumps in the pHi trace were elicited by the stepwise removal of external NH4Cl (from 20 mM; described in the Materials and Methods section). Two concentrations of S20787 were tested on the pHi-dependence of βi; 10-7 and 5.10-6M. Figure 1B shows several pooled determinations of βi obtained from 12 control cells and from 6 and 7 myocytes treated with 10-7 and 5.10-6M S20787, respectively. S20787 exerted no significant effect on the pHi dependence of βi, even when used at high concentration. Therefore, the equation describing the pHi dependence of βi under control conditions (described in the Materials and Methods section) was used to estimate acid fluxes with and without S20787.
Effect of S20787 on the activity of Na+/H+ exchange
We tested the effect of 10-6M S20787 on pHi recovery after an NH4Cl (10 mM) removal induced-acid load (13) in HEPES-buffered Tyrode's solution. Because HEPES was used as extracellular buffer, pHi recovery was mainly due to Na+/H+ exchange (2)(Fig. 2A). The myocyte was subjected to two consecutive ammonium pulses: first, under control conditions and second with addition of S20787. Even at high concentration, S20787 apparently has no effect on the time course of pHi recovery in HEPES-buffered solution. Figure 2B shows the dose-response curve for S20787 tested on the acid efflux (estimated at pHi 6.9 and relative to the control efflux; n = 6 cells for each concentration tested) carried by Na+/H+ exchange. S20787 had no effect on Na+/H+ exchange, whatever the concentration applied to the myocytes (Fig. 2B).
Effect of S20787 on the Na+, HCO3--dependent transport
The experiment shown in Fig. 3A was performed to investigate the effect of S20787 on the Na+, HCO3--dependent transport in rat ventricular myocytes. This transport works in parallel with Na+/H+ exchange after an intracellular acid load (2). Therefore, S20787 (10-7M) was applied to a myocyte superfused with a medium containing both HCO3- buffer and DMA 50 μM to inhibit Na+/H+ exchange. Control pHi recovery and the recovery obtained in the presence of S20787 are superimposed in Fig. 3A to allow comparison. S20787 had no significant effect on the time course of pHi recovery under such conditions, as is further shown by Fig. 3B, which pools the average effects of different concentrations of S20787 (range 10-11-10-6M) on the acid efflux carried by the Na+, HCO3--dependent transport (estimated at pHi 6.85 and relative to the control efflux; n = 6 cells for each concentration tested). Even at 10-6M, this drug did not significantly change the acid efflux proceeding through the Na+, HCO3--dependent transport. pHi recovery on application of NH4Cl (i.e., after intracellular alkalinization) was significantly slowed in the presence of S20787 (mean acid influx JH1 = 2.17 ± 0.1 mEq/L/min, n = 6, control, vs. 1.47 ± 0.3 mEq/L/min, n = 6, in the presence of S20787 at pHi 7.15; p < 0.05, t test), which appears to indicate an effect on Cl-/HCO3- exchange (Fig. 3A).
Effect of S20787 on the activity of Cl-/HCO3- exchange
Figure 4A shows the typical time course of pHi changes after addition and subsequent removal of 40 mM Na-acetate (14) in HCO3--buffered solution recorded in the following conditions: (a) control conditions, (b) during application of S20787 (10-6M), and (c) in the presence of DIDS 200 μM (the effect of DIDS was obtained from a different myocyte). The three pHi traces are superimposed in Fig. 4A to allow comparison. An alkanline load (induced by the acetate removal) activates Cl-/HCO3- exchange in the heart (15), as was confirmed in the present study by the fact that in the presence of DIDS (which inhibits anionic transports) (16) pHi recovery from the alkalinization is greatly decreased (≈85% at the test pHi of 7.1; mean acid influx JHi = 0.49 ± 0.04 mEq/L/min, n = 3, in the presence of DIDS, vs. 3.1 ± 0.29 mEq/L/min, n = 12, control; p < 0.001, t test) (Fig. 4B). Figure 4A moreover shows that addition of 10-6M S20787 to the superfusate elicited a significant slowing of Cl-/HCO3- exchange, though less pronounced than that elicited by DIDS application.
We tested different concentrations of S20787 on the acid influx carried by Cl-/HCO3- exchange, estimated at pHi 7.1 (n = 6-7 cells for each concentration tested) (Fig. 4B). The dose-response curve clearly showed that S20787 partly inhibits the activity of Cl-/HCO3- exchange, with an IC50 of 8.8 10-10M. Moreover, the maximum inhibitory effect induced by S20787 (≈50%) was not associated with a change in the steady-state pHi [pHi = 7.05 ± 0.015 (n = 7 control cells) vs. 7.05 ± 0.01 (n = 7 cells) in the presence of 10-6M S20787]; however, pHi in treated cells was measured only 5-10 min after the drug application. Because the action mechanism of S20787 has not yet been determined, this molecule may need to accumulate in the cells to exert its effects on Cl-/HCO3- exchange and, as a result, a change in steady-state pHi could not be detected after such time.
Effect of S20787 on background acid loading
We examined the possibility that the inhibitory effect of S20787 on pHi recovery after an alkaline load would result from a reduction in background acid loading of the cells (background loading is mainly due to metabolic H+ production in rat isolated ventricular myocytes; K. Le Prigent and D. Lagadic-Gossmann, unpublished observations). As shown in Fig. 5, we detected background acid loading by inhibiting all pHi-regulating mechanisms (Na+/H+ exchange by DMA; Na+, HCO3--dependent transport and Cl-/HCO3- exchange by DIDS). In these conditions, addition of 10-6M S20787 did not significantly change the rate of intracellular acidification (n = 4 similar experiments), thus indicating a direct effect of the drug on Cl-/HCO3- exchange.
In the present study, we tested the effect of S20787 on the three pHi-regulating membrane mechanisms identified so far in rat isolated ventricular myocytes: the dual acid extrusion system, i.e., Na+/H+ exchange and the Na+, HCO3--dependent transport and Cl-/HCO3- exchange. Our data show that: (a) a high dose (5 × 10-6M) of S20787 does not change intracellular buffering power, βi; (b) the dual acid extrusion system is not affected by S20787 in the concentration range of 10-11-10-6M; and (c) S20787 partially inhibits (≈50%) the activity of Cl-/HCO3- exchange in a dose-dependent manner, with an IC50 of 8 × 8 10-10M. Therefore, our results demonstrate that S20787 is a specific and potent partial inhibitor of Cl-/HCO3- exchange in cardiac cells.
After an intracellular alkalinization, pHi recovery was significantly slowed by S20787. A decrease in the cell H+ production through an effect of the drug on metabolism may explain this slowing. However, that S20787 remains ineffective on background acid loading strongly supports the assumption of an effect of S20787 on the anion exchange activity. Whether S20787 acts directly on Cl-/HCO3- exchange or affects some cellular processes involved in the basal modulation of this exchange is not known. With respect to the latter hypothesis, one could presume changes, e.g., in intracellular second messenger status, may have affected all three pHi-regulating mechanisms (17,18), which was clearly not so.
Until now, the most commonly used inhibitors of Cl-/HCO3- exchange have been the disulfonic stilbene derivatives [such as SITS (4-acetamido-4′-isothiocyano-2,2′-disulfonic stilbene) or DIDS]. However, these inhibitors are not specific for this exchange since they inhibit all anion permeabilities (16). Consequently, determining the relative contribution of Cl-/HCO3- exchange and of the Na+, HCO3--dependent transport in the maintenance of steady-state pHi has not yet been possible in cardiac cells. Therefore, further work is now required to determine the action mechanism of S20787 on Cl-/HCO3- exchange (i.e., the activity/structure relationship), which might help elaborate new specific and more potent inhibitors of the two HCO3--dependent, pHi-regulating mechanisms.
Cells rarely encounter conditions that promote cytoplasmic alkalosis; consequently, little attention has been accorded to the mechanisms that guard against an increase in pHi. Nevertheless, dangerous increases in pHi can occur during respiratory alkalosis, in which the pCO2 of the blood is reduced by hyperventilation, or during metabolic alkalosis, induced by an intake of excess HCO3-. In the heart, Cl-/HCO3- exchange is the sole mechanism responsible for pHi recovery from alkalosis (3). Recent work by Desilet and colleagues showed that this exchange can be modulated by different agonists, e.g., extracellular MgATP (19) and isoprenaline (20). In pathological conditions, such as ischemia, maximal activation of Cl-/HCO3- exchange by extracellular MgATP would induce an intracellular acidification that could be additive to the metabolic acidification already existent in ischemia. Furthermore, this acidification has been proposed to displace intracellular Ca2+ ions and open a nonspecific conductance, which in turn could lead to arrhythmia observed in these pathological conditions (21). In this context, specific inhibition of Cl-/HCO3- exchange by S20787 might serve to reduce not only the amplitude of the intracellular acidification but also the appearance of arrhythmia in ischemia. Further research is now needed to seek putative antiishemic properties of S20787.
Acknowledgment: This work was supported by the Institut de Recherches Internationales Servier, France.
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