Abstract
With the help of single-cell microflorimetry, 45Ca2+ radiotracer fluxes, and patch-clamp in whole-cell configuration, we examined the effect of the amiloride derivative 3-amino-6-chloro-5-[(4-chloro-benzyl)amino]-N-[[(2,4-dimethylbenzyl)amino]iminomethyl]-pyrazinecarboxamide (CB-DMB) on the activity of the three isoforms of the Na+/Ca2+ exchanger (NCX) and on several other membrane currents including voltage- and pH-sensitive ones. This amiloride analog suppressed the bidirectional activity of all NCX isoforms in a concentration-dependent manner. The IC50 values of CB-DMB were in the nanomolar range for the outward and the inward components of the bidirectional NCX1, NCX2, and NCX3 activity. Deletion mutagenesis showed that CB-DMB inhibited NCX activity mainly at level of the f-loop but not through the interaction with Gly833 located at the level of the α2 repeat. On the other hand, CB-DMB suppressed in the micromolar range the other plasma membrane currents encoded by voltage-dependent Ca2+ channels, tetrodotoxin-sensitive Na+ channels, and pH-sensitive ASIC1a. Collectively, the data of the present study showed that CB-DMB, when used in the nanomolar range, is one of the most potent compounds that can block the activity of the three NCX isoforms when they work both in the forward and in the reverse modes of operation without interfering with other ionic channels.
- CB-DMB, 3-amino-6-chloro-5-[(4-chloro-benzyl)amino]-N-[[(2,4-dimethylbenzyl)amino] iminomethyl]-pyrazinecarboxamide
- DMSO, dimethyl sulfoxide
- MES, 4-morpholineethanesulfonic acid
- MTT, 3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide
- NCX, Na+-Ca2+ exchanger
- [Ca2+]i, intracellular calcium concentration
- BHK, baby kidney hamster
- ENaC, epithelial Na+ channel
- NHE, Na+/H+ exchanger
- TEA, tetraethylammonium
- TTX, tetrodotoxin
- VDCC, voltage-dependent calcium channel
- SN6, 2-[4-(4-nitrobenzyloxy)benzyl]thiazolidine-4-carboxylic acid ethyl ester
- KB-R7943, 2-[2-[4-(4-nitrobenzyloxyl)phenyl]ethyl]isothiourea methanesulfonate
- SEA0400, 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline
- NMDG, N-methyl-d-glucamine
- YM-244769, N-(3-aminobenzyl)-6-{4-[(3-fluorobenzyl)oxy]-phenoxy}nicotinamide.
More than 40 years ago, amiloride and amiloride analogs were described by Cragoe and colleagues (1967), via a biological screen process, as potent inhibitors of sodium channels in urinary epithelium (ENaC), thus acting as a potassium-sparing diuretic. Amiloride and its analogs were subsequently shown to be inhibitors of other membrane transporters. In the same year that amiloride was synthesized, Baker and Blaustein (1968) functionally discovered the existence of the ubiquitous plasma membrane sodium/calcium exchanger (NCX). Amiloride, at very high concentrations, was found to be an effective inhibitor of NCX. Since then, amiloride has been used by numerous investigators as a probe to block NCX function (Sharikabad et al., 1997). Unfortunately, two major drawbacks have limited its use. First, millimolar concentrations are required for its NCX inhibitory activity; second, it lacks specificity, for it can also inhibit both the ENaC at micromolar concentrations (Teiwes and Toto, 2007) and the Na+/H+ exchanger (NHE) in the millimolar range. In the effort to develop amiloride derivatives provided with greater selectivity, Cragoe synthesized compounds more specific for NCX and NHE, respectively. In particular, two classes of amiloride analogs have been developed. The amiloride analogs of the first class, such as 5-[N-methyl-N-(guanidinocarbonylmethyl)] amiloride, bear substituents on the 5-amino nitrogen atom of the pyrazine ring (Taglialatela et al., 1988). They lack inhibitory properties on ENaC and NCX, even though they display greater effectiveness in inhibiting NHE in the 1 to 10 μM range (Amoroso et al., 1990; Taglialatela et al., 1990; Watano et al., 1996). The compounds of the second class, having no inhibitory effect on the Na+/H+ exchanger, bear substituents on the terminal guanidino nitrogen atom and behave as specific inhibitors (Ki 1–10 μM) of ENaC and NCX.
Among these compounds, CB-DMB seems to be the most specific inhibitor of NCX activity, because it has no inhibitory properties against NHE and the ENaC in excitable cells (Sharikabad et al., 1997), such as neurons, in which the kidney ENaC are not expressed (Taglialatela et al., 1988, 1990). On the other hand, during the past 10 years, the isothiourea derivative, KB-R7943, gained the reputation as a specific compound for inhibiting the antiporter and therefore as a probe to test the functional role of NCX . It has been reported that KB-R7943, at low micromolar concentrations, blocks the reverse mode operation of NCX, whereas high micromolar concentrations are needed to inhibit the forward mode (Iwamoto et al., 1996; Sobolevsky and Khodorov, 1999). However, recent reports have obscured its reputation as a selective probe for NCX inhibition. In fact, several studies showed that KB-R7943 also exerts an inhibitory effect on several other pharmacological targets such as NHE, dihydropyridine-sensitive Ca2+ uptake, passive Na+ uptake, Ca2+-ATPases, and Na+,K+-ATPase (Watano et al., 1996) and receptor-operated N-methyl-d-aspartate channels (Sobolevsky and Khodorov, 1999). Recently, it was reported that the NCX inhibitor KB-R7943 activates large-conductance Ca2+-activated K+ channels in endothelial and vascular smooth muscle cells (Liang et al., 2008). However, in the last decade several studies described CB-DMB as an effective probe to study the role of NCX in some pathophysiological mechanisms, including platelet hyperactivity in diabetes (Li et al., 2001), collagen-induced platelet activation (Li et al., 2001), and in vitro and in vivo ischemic cell death mechanisms (Amoroso et al., 2000; Pignataro et al., 2004; Secondo et al., 2007; Tortiglione et al., 2007; Formisano et al., 2008). Nevertheless, poor information is presently available about the effect of those compounds on each NCX isoform. In the present article, with the help of a more simple synthetic procedure that gives an increased yield and purification degree of CB-DMB, we investigated by means of patch-clamp technique, single-cell Fura-2 microfluorimetry, 45Ca2+ radiotracer fluxes, and deletion mutagenesis: 1) the specificity of CB-DMB on each of the three gene products NCX1, NCX2, and NCX3 in stably transfected baby hamster kidney (BHK) cells; 2) the selectivity of CB-DMB inhibition on the reverse and the forward modes of operation of NCX1, NCX2, and NCX3; 3) the molecular determinants of NCX molecule for CB-DMB action on each isoforms; 4) the possible interference of CB-DMB on other ionic channels such as voltage-dependent tetrodotoxin (TTX)-sensitive Na+, L-type Ca2+, and ASIC1a currents.
Materials and Methods
Drugs and Chemicals.
All chemicals used for the synthesis of CB-DMB were purchased from commercial sources.
Chemistry.
The course of reactions and purity of products were controlled by TLC (silica gel 60F254s; Merck, Darmstadt, Germany) and spots were detected by UV radiation and exposition to iodine. Flash chromatography was performed on Merck silica gel (0.040–0.063 mm). Melting points were determined with a Kofler hot-stage microscope (Leica VM HB Hotbench; Leica, Milan, Italy) and are uncorrected. Elemental analyses, indicated by the symbols of the elements, were within ± 0.4% of theoretical values. Electrospray ionization mass spectra were recorded with an API 2000 liquid chromatography/tandem mass spectrometry system (Applied Biosystems, Foster City, CA). Proton and carbon-13 nuclear magnetic resonance (NMR) spectra were recorded on a Mercury 400 spectrometer (Varian, Inc., Palo Alto, CA) operating at 400 MHz. Chemical shift values are reported in δ units (ppm) relative to tetramethylsilane used as the internal standard.
Synthesis of N-(2,4-Dimethylbenzyl)guanidine (a).
An aqueous solution of S-methylisothiourea hemisulfate salt (2.06 g; 14.8 mmol) was added to an ethanolic solution of 2,4-dimethylbenzylamine (2 g; 14.8 mmol). The mixture was refluxed for 2 h. After evaporation of ethanol, the title compound precipitated from water as sulfate salt. The solid was collected by filtration and dried. Under anhydrous conditions, Na (250 mg) was dissolved in dry methanol (20 ml) and the resulting solution was treated with dry N-(2,4-dimethylbenzyl)guanidine sulfate and stirred for 45 min. The Na2SO4 formed was removed by filtration, and the methanolic solution was evaporated in a vacuum, yielding the title compound as a yellow solid (1.44 g; 55%); m.p. 74 to 75°C. 1H NMR (CD3OD): δ 2.27, 2.28 (2s, 6H, –CH3); 4.30 (s, 2H, –CH2); 4.65 (br s, guanidine); 6.99 (d, 1H, 6-H); 7.03 (s, 1H, 3-H); 7.12 (d, 1H, 5-H); 13C NMR (CD3OD): δ 22 and 24 (2CH3); 47 (CH2); 130.06 (C-6); 131.02 (C-5); 135 (C-3); 135.02 (C-4); 140 (C-2); 141.07 (C-1); 162 (C-guanidine). m/z: 178 (M + H)+. Anal. (C10H15N3) C, H, N.
Synthesis of Methyl 3-Amino-5-(4-chloro-benzyl)amino-6-chloropyrazine-2-carboxilate (b).
A mixture of methyl 3,5-diamino-6-chloropyrazine-2-carboxylate (2 g; 9.86 mmol), 4-chlorobenzylbromide (3.04 g; 14.78 mmol), K2CO3 (2.73 g; 19.7 mmol), and dry acetone (50 ml) was refluxed for 2 h. Evaporation of the solvent gave a residue that was taken up in ethyl acetate and washed with water. The organic layer was dried on Na2SO4, which was filtered and then evaporated in a vacuum. The obtained residue, which underwent flash chromatography by elution with CHCl3/n-hexane 9:1, yielded the title compound as a yellow solid (1.60 g; 50%); m.p. 144 to 145°C. 1H NMR (DMSO-d6): δ 3.85 (s, 3H, –OCH3); 4.62 (s, 2H, –CH2); 7.20 (s, 2H, 3-NH2) 7.35, (m, 4H, 2′-H, 3′-H, 5′-H, 6′-H); 8.20 (s, 1H, 5-NH). 13C NMR (DMSO-d6): δ 43.7 (CH2); 52 (OCH3); 109.3 (C-2); 120.5 (C-6); 128.8 (C-5′ and C-3′); 130.2 (C-2′ and C-6′); 132.1 (C-4′); 138.4 (C-1′); 151.8 (C-5); 156 (C-3); 166.5 (CO). m/z: 327 (M + H)+. Anal. (C13H12Cl2N4O2) C, H, Cl, N.
Synthesis of CB-DMB.
A mixture of a (1 g; 5.5 mmol) and b (925 mg; 2.8 mmol), dissolved in dry methanol, was refluxed for 4 h. Evaporation of the solvent gave a residue that underwent flash chromatography by elution with CHCl3/MeOH 95:5. The titled compound was crystallized from diethyl ether as a yellow solid (500 mg, 38%); m.p. 153 to 154°C. 1H NMR (CD3OD): δ 2.27, 2.28 (2s, 6H, –CH3); 4.40 (s, 2H, –CH2′); 4.62 (s, 2H, –CH2″); 6.99 (d, 1H, 6′-H); 7.03 (s, 1H, 3′-H); 7.12 (d, 1H, 5′-H); 7.18 (br s, guanidine); 7.20 (s, 2H, 3-NH2) 7.32 (d, 2H, 3″-H, 5″-H), 7.35 (d, 2H, 2″-H, 6″-H); 8.20 (s, 1H, 5-NH). 13C NMR (CD3OD): δ 18 and 20 (2CH3); 43 (CH2″); 44 (CH2′); 111 (C-2); 120.5 (C-6); 126.8 (C-3″); 127 (C-6′); 128 (C-5′); 128.8 (C-5″); 129.2 (C-6″); 131.1 (C-3′); 131.2 (C-2″); 132.3 (C-4′); 132.5 (C-4″); 136.1 (C-2′); 137.9 (C-1″); 138.4 (C-1′); 151.8 (C-5); 156 (C-3); 156 (C-guanidine); 168 (CO). m/z: 472. Anal. (C22H23Cl2N7O) C, H, Cl, N.
Cell Culture.
Stably transfected BHK cells with canine cardiac NCX1, rat brain NCX2, or NCX3 were grown on plastic dishes in a mix of Dulbecco's modified Eagle's medium and Ham's F12 medium (1:1) (Invitrogen, Milan, Italy) supplemented with 5% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO). Pituitary GH3 cells were obtained from Flow Laboratories (Irvine, Scotland) and grown on plastic dishes in Ham's F10 medium (Invitrogen) composed of 15% horse serum (Flow Laboratories), 2.5% fetal calf serum (Hyclone Laboratories, Logan, UT), 100 I.U. penicillin/ml, and 100 μg streptomycin/ml. Human Embryonic Kidney (HEK293) cells were cultured in modified Eagle's medium with Earle's salts, supplemented with 10% fetal calf serum, nonessential amino acids, and glutamine. All the cells were cultured in a humidified 5% CO2 atmosphere, and the culture medium was changed every 2 days. For microfluorimetric and electrophysiological studies, cells were seeded on glass coverslips (Thermo Fisher Scientific, Waltham, MA) coated with poly(l-lysine) (30 μg/ml) (Sigma-Aldrich) and used at least 12 h after seeding.
Generation and Stable Expression of Wild-type and Mutant Exchangers.
Dog heart NCX1.1, rat brain NCX2.1, and rat brain NCX3.3 cDNAs were cloned into pcDNA3.1 expression vector. NCX1, NCX2, and NCX3 mutants were generated by means of QuikChange Site-Directed Mutagenesis Kit (Stratagene, Milan, Italy). Amino acid regions 241 to 680, 275 to 667, and 292 to 708 were deleted from NCX1, NCX2, and NCX3, respectively. G833C in NCX1 was also obtained by means of the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Wild-type and mutant exchangers were transfected in BHK cell line by Lipofectamine 2000 (Invitrogen) protocol. Stable cell lines were selected by G418 resistance and by means of the calcium-killing procedure with the Ca2+ ionophore ionomycin. In the presence of this ionophore, cells not expressing the exchanger died of Ca2+ overload.
[Ca2+]i Measurement.
[Ca2+]i was measured by single-cell, computer-assisted videoimaging (Secondo et al., 2000; 2007). In brief, BHK cells, grown on glass coverslips, were loaded with 6 μM Fura-2 acetoxymethyl ester (Fura-2AM) for 30 min at 37°C in normal Krebs' solution containing the following: 5.5 mM KCl, 160 mM NaCl, 1.2 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose, and 10 mM HEPES-NaOH, pH 7.4. At the end of the Fura-2AM-loading period, the coverslips were placed in a perfusion chamber (Medical Systems Corp., Greenvale, NY) mounted onto the stage of an inverted Axiovert 200 microscope (Carl Zeiss, Jena, Germany) equipped with a Fluar 40× oil objective lens (Carl Zeiss). The experiments were conducted with a digital imaging system composed of MicroMax 512BFT cooled CCD camera (Princeton Instruments, Trenton, NJ), LAMBDA 10-2 filter wheeler (Sutter Instrument Company, Novato, CA), and Meta-Morph/MetaFluor Imaging System software (Universal Imaging, West Chester, PA). After loading, cells were alternatively illuminated at wavelengths of 340 nm and 380 nm by a Xenon lamp. The emitted light was passed through a 512-nm barrier filter. Fura-2 fluorescence intensity was measured every 3 s. Forty to sixty-five individual cells were selected and monitored simultaneously from each cover slip. All the results are presented as cytosolic Ca2+ concentration. Assuming that the KD for Fura-2 was 224 nM, the equation of Grynkiewicz et al. (1985) was used for calibration. NCX activity, shown in Fig. 1, was evaluated as Ca2+ uptake through the reverse mode by switching the normal Krebs' medium to Na+-deficient NMDG+ medium (Na+-free): 5.5 mM KCl, 147 mM N-methylglucamine, 1.2 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose, and 10 mM HEPES-NaOH, pH 7.4 (Secondo et al., 2007). CB-DMB was incubated for 30 min before studying NCX activity.
Measurement of Na+-Dependent 45Ca2+ Uptake and 45Ca2+ Efflux.
45Ca2+ influx into the cells was measured by the method described previously (Secondo et al., 2007). After treatments, cells cultured in 24-well dishes were incubated in normal Krebs' medium: 5.5 mM KCl, 145 mM NaCl, 1.2 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose, and 10 mM HEPES-NaOH, pH 7.4, containing 1 mM ouabain and 10 μM monensin at 37°C for 10 min. Then, 45Ca2+ uptake was initiated by switching the normal Krebs' medium to Na+-free NMDG: 5.5 mM KCl, 147 mM N-methyl-d-glucamine (NMDG), 1.2 mM MgCl2, 1.5 mM CaCl2, 10 mM glucose, and 10 mM HEPES-NaOH, pH 7.4, containing 10 μM 45Ca2+ (74 kBq/ml) and 1 mM ouabain. After 30 s of incubation, cells were washed with an ice-cold solution containing 2 mM La3+ to stop 45Ca2+ uptake. Cells were subsequently solubilized with 0.1 N NaOH and aliquots were taken to determine radioactivity and protein content (Bradford, 1976).
To measure 45Ca2+ efflux, cells were loaded with 1 μM 45Ca2+ (74 kBq/ml) together with 1 μM ionomycin for 60 s in normal Krebs' solution. Next, cells were exposed either to a Ca2+- or a Na+-free solution—a condition that blocks both intracellular 45Ca2+ efflux and extracellular Ca2+ influx—or to a Ca2+-free plus 2 mM EGTA containing 145 mM Na+, a condition that promotes 45Ca2+ efflux. Thapsigargin (1 μM) was present in both solutions. 45Ca2+ efflux was started by use of Ca2+-free, Na+-containing solution plus 2 mM EGTA. Cells were exposed to this solution, which promotes 45Ca2+ efflux, for 10 s. At the time chosen (10 s), a very low efflux was detected in BHK wild-type cells. Na0+-dependent 45Ca2+ efflux was estimated by subtracting 45Ca2+ efflux in Ca2+- and Na+-free from that in Ca2+-free solution. Cells were subsequently solubilized with 0.1 N NaOH, and aliquots were taken to determine radioactivity and protein content by the Bradford method (Bradford, 1976). CB-DMB was incubated for 30 min before studying NCX activity.
Electrophysiology.
NCX currents (INCX) was recorded from BHK wild-type (Wt) and BHK-NCX1, BHK-NCX2, and BHK-NCX3 stably transfected cells by patch-clamp technique in whole-cell configuration, as described previously (Molinaro et al., 2008). Currents were filtered at 5 kHz and digitized by use of a Digidata 1322A interface (Molecular Devices, Sunnyvale, CA). Data were acquired and analyzed by use of pClamp software (version 9.0, Molecular Devices). In brief, INCX was recorded starting from a holding potential of −60 mV up to a short-step depolarization at +60 mV (60 ms) (He et al., 2003). Then, a descending voltage ramp from +60 mV to −120 mV was applied. The current recorded in the descending portion of the ramp (from +60 to −120 mV) was used to plot the current-voltage (I–V) relation curve. The magnitudes of INCX were measured at the end of +60 mV (reverse mode) and at the end of −120 mV (forward mode), respectively. The Ni2+-insensitive components were subtracted from total currents to isolate INCX (Molinaro et al., 2008). External Ringer's solution contained 126 mM NaCl, 1.2 mM NaHPO4, 2.4 mM KCl, 2.4 mM CaCl2, 1.2 mM MgCl2, 10 mM glucose, 18 mM NaHCO3, 20 mM tetraethylammonium (TEA), 10 nM TTX, and 10 μM nimodipine, pH 7.4. The dialyzing pipette solution contained 100 mM potassium gluconate, 10 mM TEA, 20 mM NaCl, 1 mM Mg-ATP, 0.1 mM CaCl2, 2 mM MgCl2, 0.75 mM EGTA, 10 mM HEPES, adjusted to pH 7.2 with CsOH. TEA (20 mM) and Cs were included to block delayed outward rectifier K+ components, nimodipine (10 μM) and TTX (50 nM) were added to external solution to block L-type Ca channels and TTX-sensitive Na+ channels, respectively. INCX values were normalized for membrane capacitance as reported previously (Molinaro et al., 2008).
Ca2+ current was recorded from pituitary GH3 cells by the patch-clamp technique in whole-cell configuration (Secondo et al., 2000). Cells were perfused with an extracellular solution containing 10 mM BaCl2, 125 mM NaCl, 1 mM MgCl2, 10 mM HEPES, and 50 nM TTX, pH 7.3. The pipettes were filled with 110 mM CsCl, 10 mM TEA, 2 mM MgCl2, 10 mM EGTA, 8 mM glucose, 2 mM Mg-ATP, 0.25 mM cAMP, and 10 mM HEPES, pH 7.3. The Ba2+ currents through Ca2+ channels were obtained by subtracting the current elicited in the presence of 50 μM CdSO4. Ba2+ currents flowing through VDCC were activated by test pulses from −60 to 0 mV (100-ms duration) from holding of −60 mV, elicited at 0.066 Hz frequency (1 pulse every 15 s). The amplitude of the Ba2+ currents was measured at the end of each depolarizing pulse.
For TTX-sensitive Na+ channel recordings, pituitary GH3 cells were perfused with an extracellular Ringer's solution containing 20 mM TEA and 5 μM nimodipine. The pipettes were filled with 110 mM CsCl, 10 mM TEA, 2 mM MgCl2, 10 mM EGTA, 8 mM glucose, 2 mM Mg-ATP, 0.25 mM cAMP, and 10 mM HEPES, pH 7.3. TTX-sensitive Na+ currents were recorded by applying, from an holding potential of −70 mV, depolarizing voltage steps of 50-ms duration in 10 mV from −100 to + 50 mV elicited at 0.066-Hz frequency (1 pulse every 15 s). The current-voltage relationship of Na+ currents were obtained normalizing the peak value for the membrane voltage imposed during the step.
ASIC1a currents were recorded with whole-cell patch-clamp technique as reported previously (Gunthorpe et al., 2001). The normal extracellular solutions contained 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, pH 7.4, and pH was adjusted by use of NaOH or HCl. For solution with pH 6.0, 10 mM glycine and MES instead of HEPES were used for more reliable pH buffering. The pipettes were filled with 30 mM NaCl, 120 mM KCl, 2 mM MgCl2, 10 mM HEPES with 300 mOsm. A multibarrel perfusion system (SF-77B, Warner Instruments, Hamden, CT) was used for rapid exchange of solutions. Data were acquired by an Axopatch 200B amplifier and analyzed by use of the pClamp software (version 10.0, Molecular Devices). Traces were filtered at 5 kHz and digitized using a Digidata 1322A interface (Molecular Devices). To obtain the IC50 of CB-DMB on each aforementioned current, all data were fitted to the following binding isotherm: y = max/{1 + (X/IC50)}n, where X is the drug concentration and n is the Hill coefficient.
Determination of Mitochondrial Activity.
Mitochondrial dysfunction was evaluated with (3[4,5-dimethylthiazol-2-y1]-2,5-diphenyltetrazolium bromide, MTT) test (Holt et al., 1987; Amoroso et al., 1999). In brief, after the experimental procedures, BHK-Wt, BHK-NCX1, BHK-NCX2, and BHK-NCX3 cells were washed with normal Krebs' solution and incubated with 1 ml of MTT solution (0.5 mg/ml in phosphate-buffered saline). This yellow water-soluble tetrazolium salt is converted into a water-insoluble purple formazan by the succinate dehydrogenase system of the active mitochondria. Therefore, the amount of formazan produced is proportional to the number of cells with mitochondria that are still vital. After 1 h of incubation at 37°C, cells were dissolved in 1 ml of DMSO, in which the rate of MTT reduction was measured by a spectrophotometer at a wavelength of 540 nm. Data are expressed as percentage of mitochondrial dysfunction versus sham-treated cultures.
Statistical Analysis.
Data are expressed as mean (± S.E.M.). Statistical comparisons between controls and treated experimental groups were performed by use of the one-way analysis of variance, followed by Newman-Keuls test. P < 0.05 was considered statistically significant.
Results
Effect of CB-DMB on NCX1, NCX2, and NCX3 Activity Evaluated as Na+-Dependent [Ca2+]i Increase, Na0+-Dependent 45Ca2+ Efflux, Nai+-Dependent 45Ca2+ Uptake and by Patch-Clamp Technique in Stably Transfected BHK-NCX1, BHK-NCX2, and BHK-NCX3 Cells.
To evaluate the inhibitory profile of CB-DMB on each isoform of the exchanger, NCX1, NCX2 and NCX3 activity was assessed as Na+ gradient-dependent [Ca2+]i increase in stably transfected BHK-NCX1, BHK-NCX2, and BHK-NCX3 cells, each expressing only the respective NCX isoforms (Secondo et al., 2007). In particular, NCX activity was evaluated in the reverse mode of operation elicited by a single pulse of Na+-deficient NMDG+ medium (Na+-free) perfused in the presence of thapsigargin (1 μM), a specific and irreversible inhibitor of the sarco(endo)plasmic reticulum (SERCA). In BHK-NCX1, BHK-NCX2, and BHK-NCX3, Na+-free perfusion caused a rapid linear rise in [Ca2+]i that was reduced by CB-DMB in a dose-dependent way (0.1–10 μM) with the following IC50 values: 286 nM for NCX1, 241 nM for NCX2, and 177 nM for NCX3 (Fig. 1). As a matter of course, Na+-free solution failed to produce a rise in [Ca2+]i in wild-type BHK cells (BHK-Wt), which lacked all three NCX isoforms, whereas ionomycin (1 μM) produced a rapid increase in [Ca2+]i (Fig. 1 A).
The effect of CB-DMB on each modality of NCX transport was also determined in BHK-NCX1, BHK-NCX2, and BHK-NCX3 cells by means of radiotracer fluxes as Na0+-dependent 45Ca2+ efflux and Nai+-dependent 45Ca2+ uptake in the presence of different concentrations of the amiloride analog (0.1–10 μM) (Fig. 2). CB-DMB was able to reduce both the forward and the reverse modes of operation of NCX1, NCX2, and NCX3 in dose-dependent manner with the following IC50 values: 550 nM for NCX1 forward, 390 nM for NCX1 reverse; 700 nM for NCX2 forward, 490 nM for NCX2 reverse; and 600 nM for NCX3 forward, 420 nM for NCX3 reverse.
The effect of CB-DMB on NCX1, NCX2, and NCX3 activity was assessed more directly by the patch-clamp technique in whole-cell configuration in stably transfected BHK cells. The whole-cell current was measured at +60 mV and −120mV by use of the ramp-clamp protocol (Materials and Methods). To isolate INCX, cells were recorded for 5 min with the well known NCX inhibitor NiCl2 (5 mM). The Ni2+-sensitive component, representing the isolated INCX, was obtained as the difference between the Ni2+-sensitive and -insensitive components (Fig. 3,B–G). No current corresponding to INCX was recorded in BHK-Wt cells (Fig. 3A) and no effect was detected after Ni2+ application (Fig. 3A). The incubation with CB-DMB (0.1–10 μM) strongly inhibited both the outward (reverse mode) and inward (forward mode) direction of INCX1, INCX2, and INCX3 in dose-dependent way with the following IC50 values: 300 nM for NCX1 forward, 320 nM for NCX1 reverse; 300 nM for NCX2 forward, 250 nM for NCX2 reverse; and 260 nM for NCX3 forward, 230 nM for NCX3 reverse (Fig. 3).
Effect of CB-DMB in NCX1, NCX2, and NCX3 Mutants Lacking “f-Loop” in Stably Transfected BHK Cells.
To characterize the interaction of CB-DMB with the molecular structure of each NCX isoform, the intracellular “f-loop,” a region mainly involved in the regulation of NCX function, has been mutated in NCX1, NCX2, and NCX3 genes by means of the deletion mutagenesis (Materials and Methods). Specifically, the effect of the amiloride analog was investigated in NCX1 Δ241–680, NCX2 Δ263–668, and NCX3 Δ292–708 mutants, whose activity, reported as percentage of the respective wild-type isoforms, was not significantly different from that of the corresponding wild-type isoforms (i.e., NCX1 Δ241–680 111% ± 10 versus NCX1 Wt; NCX2 Δ263–668 98.5% ± 8 versus NCX2 Wt; NCX3 Δ292–708 92.4% ± 12 versus NCX3 Wt). In these mutant cells, in which most of the hydrophilic domain of NCXs was deleted, both the Na+-free-induced rise in [Ca2+]i and INCX recorded in forward and reverse modes of operation were inhibited at the same extent by both 1 μM and 10 μM CB-DMB (Fig. 4, A and B). This inhibition was lower if compared with the effect exerted by the drug on the activity of the wild type isoforms (Fig. 4, A and B), showing a decrease in the inhibitory efficacy of the amiloride derivative. On the other hand, the IC50 values of CB-DMB evaluated in NCX1 Δ241–680, NCX2 Δ263–668, and NCX3 Δ292–708 mutants did not significantly differ from those obtained in the corresponding wild-type isoforms (260 ± 10 nM for NCX1 Δ241–680, 400 ± 15 nM for NCX2 Δ263–668, and 350 ± 18 nM for NCX3 Δ292–708). Furthermore, deletion mutagenesis revealed that the substitution of Gly833 with cysteine in the α2 repeat of NCX1 did not prevent the inhibitory effect of CB-DMB either on Na+-free induced [Ca2+]i increase or on INCX1 (Fig. 4, C and D).
Effect of CB-DMB on L-Type VDCC in Pituitary GH3 Cells.
L-type VDCC activity was assessed in pituitary GH3 cells by patch-clamp technique in whole-cell configuration by use of the test-pulse protocol (Materials and Methods). L-type VDCC activity was recorded for 5 min in control conditions and after the preincubation with CB-DMB (0.1, 1, and 10 μM) (Fig. 5). The peak inward Ba2+ currents recorded after the treatment with the amiloride derivative was significantly inhibited compared with the respective controls. In these conditions, CB-DMB inhibited inward Ca2+ currents in dose-dependent manner with an IC50 of 2.8 μM. The entity of L-type VDCC blockade induced by the inorganic Ca2+ channel inhibitor Cd2+ (50 μM) was not significantly different from that produced by the highest concentration of CB-DMB (10 μM) (Fig. 5).
Effect of CB-DMB on TTX-Sensitive Na+ Currents in Pituitary GH3 Cells.
TTX-sensitive Na+ channel activity was assessed in GH3 cells by patch-clamp technique in whole-cell configuration by use of the test-pulse protocol (Materials and Methods). TTX-sensitive Na+-channel activity was recorded in control conditions and after the preincubation with CB-DMB (0.1–10 μM). In particular, the amiloride analog significantly inhibited TTX-sensitive Na+ currents in a dose-dependent manner (0.1–10 μM) with an IC50 of 1.6 μM (Fig. 6). CB-DMB was able to induce a significant shift of −10 mV in Na+-channel activation recorded from GH3 cells (Fig. 6C).
Effect of CB-DMB on ASIC1a Activity in HEK293 Cells.
ASIC1a activity was assessed by the patch-clamp technique in whole-cell configuration in HEK293 cells which constitutively expressed these pH-sensitive channels (Materials and Methods). Cells were clamped at holding potential of −60 mV, and ASIC1a activity was recorded at pH 6 in control conditions and in the presence of CB-DMB (1, 3, and 10 μM). Application of an acidic extracellular solution (pH 6) to HEK293 cells revealed the functional expression of an endogenous inward proton-gated current carried by ASIC1a channels (Fig. 7). Even with maintained acidification, the proton-gated current was transient in nature, because of the rapid activation and subsequent inactivation of the current. If preicubated for 30 min, the amiloride analog significantly inhibited ASIC1a currents in a dose-dependent manner with an IC50 of 2.4 μM (Fig. 7).
Effect of CB-DMB on Mitochondrial Viability in Stably Transfected BHK-NCX1, BHK-NCX2, and BHK-NCX3 Cells.
To evaluate the mitochondrial function after CB-DMB treatment, BHK-Wt-, BHK-NCX1-, BHK-NCX2-, and BHK-NCX3-transfected cells were exposed to different concentrations of CB-DBM (1, 10, and 15 μM) (Fig. 8). After 2 h of incubation with 1 and 10 μM CB-DMB, the mitochondrial activity in these clones remained unaffected (Fig. 8). At the concentration of 15 μM only, the amiloride analog produced a small, but significant, decrease in mitochondrial activity in all treated clones (Fig. 8).
Discussion
The results of the present study showed that CB-DMB is a specific inhibitor of the three antiporter isoforms because it was able to reduce, in a dose-dependent manner, NCX1, NCX2, and NCX3 activity. The IC50 values of CB-DMB, obtained by three different techniques, such as single-cell microfluorimetry, 45Ca2+ fluxes, and patch-clamp, for inhibiting NCX1, NCX2, and NCX3 isoforms, were similar and were in the nanomolar range of concentrations. In addition, the compound under study inhibited the forward and the reverse modes of operation of each isoform in the same range of nanomolar concentrations. In light of these pharmacological properties, CB-DMB can be considered a valid tool to inhibit, in those tissues that express all of the three NCX isoforms, the currents carried by the antiporter when it works in the forward or in the reverse mode of operation. The potency of CB-DMB is barely lower than that of the new isothiourea derivative YM-244769 which is the most potent available NCX1, NCX2, and NCX3 inhibitor working in the nanomolar range (Iwamoto and Kita, 2006). However, YM-244769 inhibits NCX only in the reverse mode of operation, whereas CB-DMB inhibited NCX when it acts bidirectionally. On the other hand, the benzyloxy phenyl derivative SEA0400 bidirectionally inhibits NCX1 in the nanomolar range, reduces only mildly NCX2 activity, but it does not exert any effect on NCX3 activity. The other benzyloxy phenyl derivative SN6 inhibits NCX1, NCX2, and NCX3 isoforms only in the reverse mode of operation and in the micromolar range. The widely used reference standard for inhibiting NCX, KB-R7943 displays a lower potency than CB-DMB because it inhibits the two modes of operation of NCX1, NCX2, and NCX3 with IC50 values in the micromolar range.
The use of deletion mutagenesis showed that the intracellular f-loop of the NCX molecule, which is involved in the activity regulation of the antiporter, is a relevant molecular determinant for CB-DMB inhibitory action. In fact, the removal of the f-loop in NCX1, NCX2, and NCX3 proteins caused a partial loss of drug efficacy on each mutated isoform without interfering with its IC50 values. Furthermore, this effect was more pronounced in the NCX1 mutants than in the NCX2 and NCX3 mutants. Because the lack of the f-loop did not completely remove the inhibitory action of CB-DMB, the existence of other molecular determinants that are relevant for CB-DMB effect can not be ruled out. However, the α2 repeat residue Gly833, a common molecular determinant required for other NCX1 blocker activity such as KB-R7943, SEA0400, and SN6 (Iwamoto, 2007), did not seem to be involved in CB-DMB inhibitory action because its substitution with cysteine failed to prevent the inhibitory effect exerted by the amiloride analog. This suggests that the mechanism of CB-DMB action differs from the other known NCX blockers.
Because the amiloride derivatives may interfere with other ionic channels, we investigated the effects of CB-DMB on these plasma membrane proteins in excitable cells. This amiloride derivative in concentrations effective in inhibiting NCX1, NCX2, and NCX3 currents did not exert any effect on L-type VDCC, TTX-sensitive Na+ currents, and ASIC1a. However, it should be mentioned that when CB-DMB was used at micromolar concentrations, all of these voltage- and pH-sensitive channels were also inhibited by the amiloride analog. In fact, CB-DMB IC50 values for each NCX isoform are lower than those observed for the other examined ionic channels. By contrast, other NCX inhibitors such as KB-R7943 and SEA0400, at concentrations inhibiting NCX currents, interfere with the activity of several receptors and ionic channels, including Na+/H+ exchange, dihydropyridine-sensitive Ca2+ uptake, passive Na+ uptake, Ca2+-ATPases and Na+, K+-ATPase (Watano et al., 1996; Matsuda et al., 2005), store-operated calcium channels (Arakawa et al., 2000), TRPM3,5,6 (Kraft, 2007), and receptor-operated N-methyl-d-aspartate channels (Sobolevsky and Khodorov, 1999). In addition, it has been reported recently that KB-R7943 activates large-conductance Ca2+-activated K+ channels in endothelial and vascular smooth muscle cells (Liang et al., 2008).
Collectively, the data of the present study showed that CB-DMB, when used in the nanomolar range, is one of the most potent compounds able to block the activity of the three NCX1, NCX2, and NCX3 isoforms when they work both in the forward and in the reverse modes of operation without interfering with other ionic channels.
Acknowledgments
We thank Dr. Kenneth Philipson (University of California, Los Angeles, CA) for a generous gift of dog heart NCX1.1, rat brain NCX2.1, and rat brain NCX3.3 cDNAs and Vincenzo Grillo and Carmine Capitale for technical assistance.
Footnotes
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This work was supported by COFIN 2006; the “Ministero Affari Esteri, Direzione Generale per la Promozione e la Cooperazione Culturale Fondi Italia-Cina Legge 401/1990 2007, 2008”; Ministero della Salute, Ricerca Sanitaria RF-FSL352059 Ricerca finalizzata 2006; Ministero della Salute, Ricerca Oncologica 2006; Ministero della Salute, Progetto Strategico, 2007; Ministero della Salute, Progetto Ordinario, 2007 (all to L.A.); Legge Regionale 5 28/03/2002 (to A.S.).
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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ABBREVIATIONS:
- Received February 9, 2009.
- Accepted July 13, 2009.
- © 2009 by The American Society for Pharmacology and Experimental Therapeutics