The Sodium-Calcium Antiport of Heart Mitochondria Is Not Electroneutral
1995; Elsevier BV; Volume: 270; Issue: 2 Linguagem: Inglês
10.1074/jbc.270.2.672
ISSN1083-351X
AutoresDennis W. Jung, Kemal Baysal, Gerald P. Brierley,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoHeart mitochondria contain a nNa + /Ca 2+ antiport that participates in the regulation of matrix [Ca 2+ ]. Based largely on a single study (Brand, M. D.(1985) Biochem. J. 229, 161-166), there has been a consensus that this antiport promotes the electroneutral exchange of two Na + for one Ca 2+ . However, a recent study in our laboratory (Baysal, K., Jung, D. W., Gunter, K. K., Gunter, T. P., and Brierley, G. P. (1994) Am. J. Physiol. 266, C800-C808) has shown that the Na + -dependent efflux of Ca 2+ from heart mitochondria has more energy available to it than can be supplied by a passive 2Na + /Ca 2+ exchange. We have therefore re-examined Brand's protocols using fluorescent probes to monitor matrix pH and free [Ca 2+ ]. Respiring heart mitochondria, suspended in KCl and treated with ruthenium red to block Ca 2+ influx, extrude Ca 2+ and establish a large [Ca 2+ ]out : [Ca 2+ ]matrix gradient. The extrusion of Ca 2+ under these conditions is Na + -dependent and diltiazem-sensitive and can be attributed to the nNa + /Ca 2+ antiport. Addition of nigericin increases the membrane potential (Δ Ψ ) and decreases ΔpH to 0.1 or less, but has virtually no effect on the magnitude of the [Ca 2+ ] gradient. Under these conditions a gradient maintained by electroneutral 2Na + /Ca 2+ antiport should be abolished because the mitochondrial Na + /H + antiport keeps the [Na + ] gradient equivalent to the [H + ] gradient. The [Ca 2+ ] gradient is abolished, however, when an uncoupler is added to dissipate Δ Ψ or when the exogenous electroneutral antiport BrA23187 is added. In addition, [Ca 2+ ] influx via the nNa + /Ca 2+ antiport in nonrespiring mitochondria is enhanced when Δ Ψ is abolished. These results are consistent with Ca 2+ extrusion by an electrophoretic antiport that can respond to Δ Ψ but not with an electroneutral antiport. Heart mitochondria contain a nNa + /Ca 2+ antiport that participates in the regulation of matrix [Ca 2+ ]. Based largely on a single study (Brand, M. D.(1985) Biochem. J. 229, 161-166), there has been a consensus that this antiport promotes the electroneutral exchange of two Na + for one Ca 2+ . However, a recent study in our laboratory (Baysal, K., Jung, D. W., Gunter, K. K., Gunter, T. P., and Brierley, G. P. (1994) Am. J. Physiol. 266, C800-C808) has shown that the Na + -dependent efflux of Ca 2+ from heart mitochondria has more energy available to it than can be supplied by a passive 2Na + /Ca 2+ exchange. We have therefore re-examined Brand's protocols using fluorescent probes to monitor matrix pH and free [Ca 2+ ]. Respiring heart mitochondria, suspended in KCl and treated with ruthenium red to block Ca 2+ influx, extrude Ca 2+ and establish a large [Ca 2+ ]out : [Ca 2+ ]matrix gradient. The extrusion of Ca 2+ under these conditions is Na + -dependent and diltiazem-sensitive and can be attributed to the nNa + /Ca 2+ antiport. Addition of nigericin increases the membrane potential (Δ Ψ ) and decreases ΔpH to 0.1 or less, but has virtually no effect on the magnitude of the [Ca 2+ ] gradient. Under these conditions a gradient maintained by electroneutral 2Na + /Ca 2+ antiport should be abolished because the mitochondrial Na + /H + antiport keeps the [Na + ] gradient equivalent to the [H + ] gradient. The [Ca 2+ ] gradient is abolished, however, when an uncoupler is added to dissipate Δ Ψ or when the exogenous electroneutral antiport BrA23187 is added. In addition, [Ca 2+ ] influx via the nNa + /Ca 2+ antiport in nonrespiring mitochondria is enhanced when Δ Ψ is abolished. These results are consistent with Ca 2+ extrusion by an electrophoretic antiport that can respond to Δ Ψ but not with an electroneutral antiport. INTRODUCTIONIt is now generally accepted that [Ca 2+ ] enters the matrix of heart mitochondria via the ruthenium red-sensitive uniport and is extruded on a nNa + /Ca 2+ antiport (see (1Gunter T.E. Pfeiffer D.R. Am. J. Physiol. 1990; 258: C755-C786Crossref PubMed Google Scholar) and (2Crompton M. Bronner F. Intracellular Ca Regulation. R. Liss, New York1990: 181-209Google Scholar) for reviews). Until recently there has been a consensus that this antiport promotes the electroneutral exchange of two Na + for one Ca 2+ (2Crompton M. Bronner F. Intracellular Ca Regulation. R. Liss, New York1990: 181-209Google Scholar, 3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar, 4Li W. Shariat-Madar Z. Powers M. Sun X. Lane R.D. Garlid K.D. J. Biol. Chem. 1992; 267: 17983-17989Abstract Full Text PDF PubMed Google Scholar). This conclusion is based largely on a study by Brand (3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar) that showed a [Ca 2+ ] distribution established by the endogenous antiport did not differ significantly from that produced by the ionophore A23187. Because A23187 mediates electroneutral 2H + /Ca 2+ exchange and the endogenous Na + /H + antiport equilibrates [H + ] and [Na + ] gradients, it was concluded that the nNa + /Ca 2+ antiport must be electroneutral(3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar). This stoichiometry is also supported by the more recent report of Li et al.(4Li W. Shariat-Madar Z. Powers M. Sun X. Lane R.D. Garlid K.D. J. Biol. Chem. 1992; 267: 17983-17989Abstract Full Text PDF PubMed Google Scholar) that all modes of transport promoted by a purified and reconstituted Na + /Ca 2+ antiport are insensitive to uncouplers and therefore electroneutral. However, there have also been indications that this antiport could be electrophoretic. The early report of Crompton et al.(5Crompton M. Kunzi M. Carafoli E. Eur. J. Biochem. 1977; 79: 549-558Crossref PubMed Scopus (181) Google Scholar) established that the Na + -dependent efflux of Ca 2+ was dependent on the energy state of the mitochondria and kinetic data indicate the presence of three independent Na + binding sites on the antiport(6Hayat L.H. Crompton M. Biochem. J. 1987; 244: 533-538Crossref PubMed Scopus (17) Google Scholar). These results could be explained by a contribution of the membrane potential, Δ Ψ , to an electrophoretic exchange, such as that found with the 3Na + /Ca 2+ antiport of the sarcolemma(7Pitts B.J.R. J. Biol. Chem. 1979; 254: 6232-6235Abstract Full Text PDF PubMed Google Scholar).Two recent studies from our laboratory strongly suggest that the concept of an electroneutral nNa + /Ca 2+ antiport should be re-examined. In the first it was noted that the exchanger is regulated by matrix pH and that high rates of Na + /Ca 2+ antiport can be sustained when ΔpH (and hence the Na + gradient) approaches zero(8Baysal K. Brierley G.P. Novgorodov S. Jung D.W. Arch. Biochem. Biophys. 1991; 291: 383-389Crossref PubMed Scopus (42) Google Scholar). This led us to a null-point study in which it was established that [Ca 2+ ] gradients maintained by Na + /Ca 2+ antiport are too large to be sustained by a passive electroneutral exchange of 2Na + for one Ca 2+ (9Baysal K. Jung D.W. Gunter K.K. Gunter T.E. Brierley G.P. Am. J. Physiol. 1994; 266: C800-C808Crossref PubMed Google Scholar). These observations in turn led us to the present work in which we examine the protocols of Brand (3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar) in more detail using currently available fluorescent probes to monitor [H + ] and [Ca 2+ ] gradients. In contrast to Brand(3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar), we conclude that, when the pH gradient of respiring mitochondria is collapsed by addition of nigericin, a large [Ca 2+ ]out :[Ca 2+ ]matrix gradient is maintained. Addition of an uncoupler to dissipate Δ Ψ abolishes this gradient, as does addition of the electroneutral exchanger BrA23187. These experiments lead to the conclusion that the nNa + /Ca 2+ antiport is not electroneutral under these conditions and that there is a large contribution of Δ Ψ to the observed [Ca 2+ ] gradient. Experiments showing Na + -dependent [Ca 2+ ] influx in nonrespiring mitochondria also support the conclusion that the mitochondrial nNa + /Ca 2+ antiport is electrophoretic.EXPERIMENTAL PROCEDURESEquilibration of Mitochondria with Fluorescent ProbesBeef heart mitochondria were prepared as described previously (10Brierley G.P. Jurkowitz M.S. Farooqui T. Jung D.W. J. Biol. Chem. 1984; 259: 14672-14678Abstract Full Text PDF PubMed Google Scholar) and suspended at 25 mg/ml in sucrose (0.25 M), containing TES 1The abbreviations used are as follows: TES2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acidBCECF2′,7′-bis(carboxyethyl)-5(6)-carboxyfluoresceinAMacetoxymethyl estercSNARF-1™a pH indicator marketed by Molecular Probes Inc.pHextramitochondrial pHpHmatrix pHΔ Ψmembrane potentialTPP+tetraphenylphosphonium ionNTAnitrilotriacetic acidFCCPcarbonyl cyanide-4-trifluoromethoxyphenylhydrazoneTMA+tetramethylammonium ion. (10 mM, K + salt, pH 7.4). Mitochondria were equilibrated with fluorescent probes as follows. Mitochondria (12.5 mg/ml) were incubated at 24°C in a medium of sucrose (0.25 M), TES (10 mM), NaCl (20 mM), ATP (1.6 mM), EGTA (0.2 mM) neutralized to pH 7.4 with KOH and containing either BCECF/AM (7.2 μM), cSNARF-1/AM (8.8 μM), SBFI/AM (11.1 μM), or fura-2/AM (5 μM). Pluronic F-127 was also added when SBFI/AM was used(14Jung D.W. Apel L.M. Brierley G.P. Am. J. Physiol. 1992; 262: C1047-C1055Crossref PubMed Google Scholar). A second suspension of mitochondria, incubated in the same medium without the fluorescent probe, was used to determine autofluorescence. After 20 min, 3 volumes of ice-cold wash medium (0.2 M sucrose, 10 mM K + TES, 30 μM EGTA, pH 7.4) were added, and the mitochondria were isolated by centrifugation and resuspended in the same medium. After an additional 5-min incubation at 24°C, the mitochondria were again diluted, reisolated by centrifugation, and suspended at 25 mg/ml in the wash medium. These loading conditions deplete the mitochondria of almost all endogenous Ca 2+ . Virtually none of the fura-2 is outside the matrix since addition of near saturating concentrations of [Ca 2+ ] to suspended mitochondria results in an insignificant increase in fura-2 fluorescence. ADP-stimulated respiration and respiratory control ratios of probe-loaded mitochondria are ~ 90% of control mitochondria(17Jung D.W. Davis M.H. Brierley G.P. Anal. Biochem. 1989; 178: 348-354Crossref PubMed Scopus (49) Google Scholar).Incubation ConditionsFor most experiments, the mitochondria containing sequestered fluorescent probes were incubated at 0.5 mg/ml in a 3-ml cuvette at 25°C in a standard medium consisting of: KCl (100 mM), HEPES (15 mM, K + salt, pH 7.35), cyclosporin A (1 μM), rotenone (1 μg/ml), and oligomycin (2 μg/ml). The EGTA carried over with the addition of mitochondria was 0.6 μM. Further additions were made as described in the legends. The experiments shown in Fig. 3 were carried out in the mannitol-sucrose medium described by Brand(3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar). [Ca 2+ ] buffers were prepared by the addition of 2 mM EGTA (for 0-2 μM [Ca 2+ ] buffers) or 2 mM NTA (5-50 μM [Ca 2+ ] buffers) and variable amounts of CaCl2 to the standard medium. The free [Ca 2+ ] for each buffer was calculated using the Fabiato computer program (11Fabiato A. Methods Enzymol. 1988; 157: 378-417Crossref PubMed Scopus (973) Google Scholar) using apparent stability constants for 25°C and pH 7.4 of 1.146 × 104 for Ca/NTA, 1.592 × 107 for Ca/EGTA, and 15.75 for Ca/succinate. The absolute log K1 for Ca/NTA was taken as 6.45 with log protonation constants of 9.71, 2.49, 1.86, and 0.8(12Anderegg G. Pure Appl. Chem. 1982; 54: 2693-2758Crossref Scopus (168) Google Scholar). The pHo was measured with a glass electrode.Fluorescence MeasurementsFluorescence was measured using a Perkin-Elmer LS-5B fluorimeter interfaced with a computer as described previously(9Baysal K. Jung D.W. Gunter K.K. Gunter T.E. Brierley G.P. Am. J. Physiol. 1994; 266: C800-C808Crossref PubMed Google Scholar, 13Jung D.W. Apel L.M. Brierley G.P. Biochemistry. 1990; 29: 4121-4128Crossref PubMed Scopus (66) Google Scholar). The monochromators were driven to obtain ratios approximately every 7 s for a single probe (13Jung D.W. Apel L.M. Brierley G.P. Biochemistry. 1990; 29: 4121-4128Crossref PubMed Scopus (66) Google Scholar, 14Jung D.W. Apel L.M. Brierley G.P. Am. J. Physiol. 1992; 262: C1047-C1055Crossref PubMed Google Scholar) or every 30 s when two probes were used(9Baysal K. Jung D.W. Gunter K.K. Gunter T.E. Brierley G.P. Am. J. Physiol. 1994; 266: C800-C808Crossref PubMed Google Scholar). BCECF fluorescence was recorded at 550 nm with excitation at 509 and 450 nm, fura-2 fluorescence at 510 nm with excitation at 340 and 365 nm, SBFI fluorescence at 510 nm with excitation at 350 and 390 nm excitation, and cSNARF-1 emission at 630 and 604 nm with excitation at 534 nm. For each experiment the exact protocol was repeated using nonloaded mitochondria to record autofluorescence. The records of autofluorescence were subtracted from the experimental records before [Ca 2+ ], [Na + ], and pHi were calculated using the ratioing procedure(8Baysal K. Brierley G.P. Novgorodov S. Jung D.W. Arch. Biochem. Biophys. 1991; 291: 383-389Crossref PubMed Scopus (42) Google Scholar, 9Baysal K. Jung D.W. Gunter K.K. Gunter T.E. Brierley G.P. Am. J. Physiol. 1994; 266: C800-C808Crossref PubMed Google Scholar, 13Jung D.W. Apel L.M. Brierley G.P. Biochemistry. 1990; 29: 4121-4128Crossref PubMed Scopus (66) Google Scholar, 14Jung D.W. Apel L.M. Brierley G.P. Am. J. Physiol. 1992; 262: C1047-C1055Crossref PubMed Google Scholar).Matrix [Ca 2+ ] was calculated after determining Rmin, Rmax, and Sf2 /Sb2 values using the following expression(15Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). [Ca2+]=Kd(R-Rmin)/(Rmax-R)Sf2/Sb2(Eq. 1) To determine Rmin, BrA23187 (2 μM) was added to respiring mitochondria suspended in the standard medium containing 2 mM EGTA to deplete mitochondrial Ca 2+ , leaving only uncomplexed fura-2 in the matrix (Fig. 1). Rmax was obtained by allowing respiring mitochondria to accumulate Ca 2+ and saturate matrix fura-2 (Fig. 1). Sf2 /Sb2 is the ratio of fluorescence intensities at 365 nm with zero and with excess Ca 2+ . Rmin, Rmax, and Sf2 /Sb2 values were determined for each mitochondrial preparation. The means ± S.E. for the experiments presented here (n = 6) were 0.626 ± 0.001 Rmin, 2.653 ± 0.113 Rmax, and 1.858 ± 0.242 Sf2 /Sb2 . The solid line in Fig. 1 represents the relationship between calculated [Ca 2+ ] and the 340/365 ratio and shows that fura-2 approaches saturation above 2 μM matrix [Ca 2+ ]. With this in mind our experiments were designed to keep matrix [Ca 2+ ] below 2 μM.Figure 1:Estimation of Rmin and Rmax for fura-2 sequestered in the matrix of heart mitochondria. Mitochondria were loaded with fura-2 as described under "Experimental Procedures" and added to the standard medium containing 2 mM EGTA for the Rmin determination and 15 μM EGTA for the Rmax determination. Succinate (5 mM) was added at 100 s. CaCl2 (2 mM) was added at 200 s for the Rmax determination and BrA23187 (2 μM) for the Rmin determination. Fluorescence was corrected for autofluorescence and ratios (340/365)ex were calculated from net fluorescence intensities. The relative net fluorescence intensities and calculated ratios were: Rmin (340/530) = 0.64, Rmax (760/254) = 3.0, and Sf2 /Sb2 (530/254) = 2.09. The solid line shows the relationship between the ratios and [Ca 2+ ] calculated according to. The initial ratio value of 1.55 indicates a matrix [Ca 2+ ] of 0.25 μM.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Estimation of Δ Ψ Membrane potential (Δ Ψ ) was estimated using [3H]TPP + distribution calibrated with 86 Rb + (16Jensen B.D. Gunter K.K. Gunter T.E. Arch. Biochem. Biophys. 1986; 248: 305-323Crossref PubMed Scopus (68) Google Scholar). Succinate respiration was inhibited with malonate to vary Δ Ψ (16Jensen B.D. Gunter K.K. Gunter T.E. Arch. Biochem. Biophys. 1986; 248: 305-323Crossref PubMed Scopus (68) Google Scholar). Mitochondrial volumes and water spaces were determined using [14C]sucrose and [3H]H2O as described previously(17Jung D.W. Davis M.H. Brierley G.P. Anal. Biochem. 1989; 178: 348-354Crossref PubMed Scopus (49) Google Scholar). Following the recent suggested convention (18Silverstein T.P. Biochim. Biophys. Acta. 1993; 1183: 1-3Crossref Scopus (34) Google Scholar), Δ Ψ and ΔpH are calculated as the difference between the matrix and medium values. This means that Δ Ψ is negative and ΔpH is positive in normal respiring mitochondria. The reader should be aware that this convention was not used by Brand (3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar) or in many of the other references cited.All chemicals used were of reagent grade purity or higher. Ruthenium red was obtained from Fluka and recrystallized according to Luft(19Luft J.H. Anat. Rec. 1971; 171: 347-368Crossref PubMed Scopus (1088) Google Scholar). The AM esters were obtained from Molecular Probes, Inc. (Eugene, OR) and were dissolved in dimethyl sulfoxide (silylation grade) obtained from Pierce.RESULTSA passive nNa + /Ca 2+ antiport will exchange Ca 2+ for Na + until the energy contained in the electrochemical gradient of Ca 2+ is balanced by the proper stoichiometric factor (n) multiplied by the energy in the electrochemical gradient of Na + (20Brand M.D. Biochem. J. 1985; 225: 413-419Crossref PubMed Scopus (25) Google Scholar, 21Gunter K.K. Zuscik M.J. Gunter T.E. J. Biol. Chem. 1991; 266: 21640-21648Abstract Full Text PDF PubMed Google Scholar, 22Crompton M. Curr. Top. Membr. Transp. 1985; 25: 231-276Crossref Scopus (87) Google Scholar). Because Na + /H + exchange is rapid in isolated mitochondria and effectively equilibrates the [Na + ] and [H + ] gradients, the following expression should hold for a passive nNa + /Ca 2+ antiport at equilibrium. Δμ~Ca2+=nΔμ~Na+=nΔμ~H+(Eq. 2) At 25°C this relationship can be expressed as follows(20Brand M.D. Biochem. J. 1985; 225: 413-419Crossref PubMed Scopus (25) Google Scholar, 21Gunter K.K. Zuscik M.J. Gunter T.E. J. Biol. Chem. 1991; 266: 21640-21648Abstract Full Text PDF PubMed Google Scholar). [Ca2+]out/[Ca2+]matrix=10((2-n)Ψ/59+nΔpH)(Eq. 3) This thermodynamic relationship predicts that when ΔpH is collapsed, no [Ca 2+ ] gradient can be maintained at equilibrium if the antiport is electroneutral (n = 2), since 10 2ΔpH = 1 under these conditions. However, a [Ca 2+ ] gradient can be maintained if the antiport promotes electrophoretic exchange (n > 2) and responds to Δ Ψ . The term "electrophoretic" is used to indicate that the transport occurs in response to an existing electrical potential. This is in contrast to a transport reaction that generates a potential that would be termed "electrogenic"(23Nicholls D.G. Ferguson S.J. Bioenergetics 2. Academic Press, London1992: 27Google Scholar).The assumption that ΔpH and ΔpNa are very near equivalent is essential to this investigation and is supported by several studies. Crompton and Heid (24Crompton M. Heid I. Eur. J. Biochem. 1978; 91: 599-608Crossref PubMed Scopus (117) Google Scholar) using isotope distribution procedures showed that the [Na + ] and [H + ] gradients are nearly at equilibrium for nonrespiring rat heart mitochondria suspended in a KCl medium. This equilibrium results from the high rate of Na + /H + exchange relative to the Na + /Ca 2+ antiport(24Crompton M. Heid I. Eur. J. Biochem. 1978; 91: 599-608Crossref PubMed Scopus (117) Google Scholar). Studies from this laboratory (14Jung D.W. Apel L.M. Brierley G.P. Am. J. Physiol. 1992; 262: C1047-C1055Crossref PubMed Google Scholar) using SBFI-equilibrated pig heart mitochondria strongly support this conclusion. When the [Na + ] gradients reported (14Jung D.W. Apel L.M. Brierley G.P. Am. J. Physiol. 1992; 262: C1047-C1055Crossref PubMed Google Scholar) are compared with the appropriate [H + ] gradients measured in related studies using the fluorescent probes BCECF (25Brierley G.P. Davis M.H. Jung D.W. Biochemistry. 1989; 28: 4347-4354Crossref PubMed Scopus (26) Google Scholar) or cSNARF-1(8Baysal K. Brierley G.P. Novgorodov S. Jung D.W. Arch. Biochem. Biophys. 1991; 291: 383-389Crossref PubMed Scopus (42) Google Scholar), the mean difference between ΔpH and ΔpNa calculates to 0.11 ± 0.06 (n = 12).Isolated beef heart mitochondria are difficult to load with SBFI (or the analogous probe Sodium Green). The fluorescence signal obtained with these preparations is no greater than 1.5 times the autofluorescence. This makes it very difficult to distinguish between changes in redox components and changes in matrix [Na + ] and has prevented us from following the [Na + ] gradient directly in the respiring beef heart mitochondria used in these studies. However, we were able to follow the spontaneous decay of the pH and pNa gradients in nonrespiring beef heart mitochondria that had been equilibrated with SBFI and cSNARF-1 (Fig. 2). In this protocol the decay of ΔpNa approximates the ΔpH decay with a maximum difference of 0.17. These results support the concept that ΔpH and ΔpNa are maintained nearly equal by the Na + /H + antiport (14Jung D.W. Apel L.M. Brierley G.P. Am. J. Physiol. 1992; 262: C1047-C1055Crossref PubMed Google Scholar, 24Crompton M. Heid I. Eur. J. Biochem. 1978; 91: 599-608Crossref PubMed Scopus (117) Google ScholarFigure 2:Spontaneous decay of ΔpH and ΔpNa in nonrespiring heart mitochondria. Mitochondria equilibrated with both SBFI and cSNARF-1 were added to the standard medium containing ruthenium red (1 μM), NaCl (19 mM), EGTA (1 mM), and CaCl2 (0.9 mM). Net fluorescence intensities were used to calculate matrix pH (8Baysal K. Brierley G.P. Novgorodov S. Jung D.W. Arch. Biochem. Biophys. 1991; 291: 383-389Crossref PubMed Scopus (42) Google Scholar) and matrix [Na + ](14Jung D.W. Apel L.M. Brierley G.P. Am. J. Physiol. 1992; 262: C1047-C1055Crossref PubMed Google Scholar). Medium pH was 7.37.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In his influential 1985 study of the stoichiometry of this antiport, Brand (3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar) used a Ca 2+ -electrode to follow medium [Ca 2+ ] in a suspension of rat heart mitochondria respiring in a low K + medium. After addition of nigericin he found the matrix to be acid with a ΔpH of about −1.0. Under these conditions he calculated that external [Ca 2+ ] for an n = 2 stoichiometry should differ from that for n = 3 by 0.634 μM at equilibrium(3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar). At equilibrium Brand (3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar) reported a ΔpH of −0.99 (using the conventions of (18Silverstein T.P. Biochim. Biophys. Acta. 1993; 1183: 1-3Crossref Scopus (34) Google Scholar)), a ΔpNa of −0.86, Δ Ψ of −161 mV, external [Ca 2+ ] of 6.37 μM, and a calculated matrix [Ca 2+ ] of nearly 500 μM for mitochondria containing approximately 3 nmol of Ca 2+ /mg of protein.These values are out of line with our experience with respiring beef heart mitochondria under quite similar conditions. Using BCECF fluorescence to monitor pHi we see a large negative ΔpH (interior acid) only for nonrespiring mitochondria following nigericin addition in the low K + medium used by Brand (Fig. 3). Under these conditions a ΔpH of about −0.8 is established immediately and decays to about −0.5 after 500 s (Fig. 3). Mitochondria respiring with succinate in the same medium show a transient ΔpH of about −0.5 following nigericin addition, but respiration rapidly establishes a condition in which ΔpH is about 0.15 (Fig. 3). This is probably the result of electrophoretic uptake of TMA + in response to the high Δ Ψ established by nigericin. Addition of succinate to nonrespiring mitochondria maintaining a large negative ΔpH results in an alkaline shift to a final ΔpH near 0.1 (Fig. 3). In the KCl medium used for the studies reported here there is no discernible ΔpH after nigericin addition whether or not respiration is taking place because nigericin equilibrates the [H + ] gradient with the [K + ] gradient(23Nicholls D.G. Ferguson S.J. Bioenergetics 2. Academic Press, London1992: 27Google Scholar). The BCECF-loaded mitochondria lose some endogenous K + during equilibration with the probe and average just over 100 ng ion K + •mg -1 protein. The incubation medium contains about 120 mM K + , so the [K + ] gradient is minimal under these conditions.These results suggest that Brand's estimate of ΔpH for respiring mitochondria treated with nigericin (3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar) is too large. The distribution of [14C]methylamine and 22 Na + was used to estimate the gradients in his study (3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar) and it is possible that either redistribution of the probe at anaerobiosis (following centrifugation), nonohmic uptake of the cationic probes, or changes in probe binding during energization in the low ionic strength suspending medium could have affected the results (see (26Jung D.W. Davis M.H. Brierley G.P. Arch. Biochem. Biophys. 1988; 263: 1928Crossref Scopus (17) Google Scholar)). At pH 7 methylamine (pKa ~ 10.5) is >99% cationic. Regardless of the reason for the discrepancy, a decrease in the ΔpNa from −0.86 as estimated by Brand (3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar) to −0.50 or less would result in loss of ability to distinguish between n = 2 and n = 3 stoichiometry in his experiment. As shown in Fig. 4 the difference in external [Ca 2+ ] predicted for these two cases becomes less than 0.1 μM at −0.5 ΔpNa. If ΔpH (and therefore ΔpNa) were as small as the experiments of Fig. 3 indicate, the difference between n = 2 and n = 3 would disappear completely (Fig. 4). If in fact the ΔpH in Brand's experiment (3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar) was much less than his estimate, then he would have seen no measurable change in external [Ca 2+ ] when A23187 was added to provide an exogenous electroneutral exchanger, even if the endogenous antiport were electrophoretic.Figure 4:Predicted values for extramitochondria [Ca 2+ ] versus the Na + gradient as a function of the stoichiometry of the nNa + /Ca 2+ antiport. Values were calculated from the data of Table I (sample 1) of (3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar) using Equation 1 in (3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar), a stoichiometry of either 2 or 3 Na + /Ca 2+ , and various values of ΔpNa. Due to uncertainties in some of the values these calculations vary slightly when compared with those of Brand(3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar). For example, our value for n = 2 and ΔpNa of −0.86 is 6.66 μM as opposed to 6.53 given in(3Brand M.D. Biochem. J. 1985; 229: 161-166Crossref PubMed Scopus (50) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Use of Fluorescent Probes to Estimate the [Ca 2+ ] Gradient Maintained by the nNa + /Ca 2+ AntiportBecause mitochondria respiring in a KCl medium do not maintain a significant pH gradient following treatment with nigericin (Fig. 5B), predicts that no [Ca 2+ ] gradient will be maintained if the stoichiometry is n = 2. On the other hand, if it is electrophoretic a [Ca 2+ ]out :[Ca 2+ ]matrix gradient of 10 (2-n)Δψ/59 will be maintained by the nNa + /Ca 2+ antiport at equilibrium.Figure 5:Effects of nigericin on matrix [Ca 2+ ] and matrix pH of respiring heart mitochondria. A, mitochondria equilibrated with fura-2 were added to the standard medium containing NaCl (20 mM), succinate (5 mM), and [Ca 2+ ] buffered at either 0.8 μM or 5 μM. The 0.8 μM [Ca 2+ ] was established by adding CaCl2 (1.86 mM) and EGTA (2 mM), whereas the 5 μM buffer contained NTA (2 mM) and CaCl2 (0.115 mM). Ruthenium red (1 μM) was added 10 s after the mitochondria and fluorescence measurements were then started. Nigericin (1 μM) was added at 100 s. B, mitochondria equilibrated with cSNARF-1 were incubated in the same medium and under the same conditions as A. Matrix pH was calculated from net fluorescence intensity as described(8Baysal K. Brierley G.P. Novgorodov S. Jung D.W. Arch. Biochem. Biophys. 1991; 291: 383-389Crossref PubMed Scopus (42) Google Scholar). The records shown are from a different mitochondrial preparation than that used in A. Records from mitochondria loaded with both fura-2 and cSNARF-1 show essentially the same picture when [Ca 2+ ] and pH are monitored simultaneously.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Heart mitochondria were equilibrated with the fluorescent pH indicator cSNARF-1 or the [Ca 2+ ] indicator fura-2 and suspe
Referência(s)