Oxygen-bridged Dinuclear Ruthenium Amine Complex Specifically Inhibits Ca2+ Uptake into Mitochondria in Vitroand in Situ in Single Cardiac Myocytes
1998; Elsevier BV; Volume: 273; Issue: 17 Linguagem: Inglês
10.1074/jbc.273.17.10223
ISSN1083-351X
AutoresMohammed A. Matlib, Zhuan Zhou, Selena Knight, Saadia Ahmed, Kin M. Choi, J.A. Krause-Bauer, Ronald M. Phillips, Ruth A. Altschuld, Yasuhiro Katsube, Nicholas Sperelakis, Donald M. Bers,
Tópico(s)Cardiac Ischemia and Reperfusion
ResumoRuthenium red is a well known inhibitor of Ca2+ uptake into mitochondria in vitro. However, its utility as an inhibitor of Ca2+ uptake into mitochondria in vivo or in situ in intact cells is limited because of its inhibitory effects on sarcoplasmic reticulum Ca2+ release channel and other cellular processes. We have synthesized a ruthenium derivative and found it to be an oxygen-bridged dinuclear ruthenium amine complex. It has the same chemical structure as Ru360 reported previously (Emerson, J., Clarke, M. J., Ying, W-L., and Sanadi, D. R. (1993) J. Am. Chem. Soc.115, 11799–11805). Ru360 has been shown to be a potent inhibitor of Ca2+-stimulated respiration of liver mitochondria in vitro. However, the specificity of Ru360 on Ca2+ uptake into mitochondria in vitro or in intact cells has not been determined. The present study reports in detail the potency, the effectiveness, and the mechanism of inhibition of mitochondrial Ca2+ uptake by Ru360 and its specificity in vitro in isolated mitochondria and in situ in isolated cardiac myocytes. Ru360 was more potent (IC50 = 0.184 nm) than ruthenium red (IC50 = 6.85 nm) in inhibiting Ca2+ uptake into mitochondria. 103Ru360 was found to bind to isolated mitochondria with high affinity (K d = 0.34 nm, B max = 80 fmol/mg of mitochondrial protein). The IC50 of 103Ru360 for the inhibition of Ca2+ uptake into mitochondria was also 0.2 nm, indicating that saturation of a specific binding site is responsible for the inhibition of Ca2+ uptake. Ru360, as high as 10 μm, produced no effect on sarcoplasmic reticulum Ca2+ uptake or release, sarcolemmal Na+/Ca2+ exchange, actomyosin ATPase activity, L-type Ca2+ channel current, cytosolic Ca2+ transients, or cell shortening. 103Ru360 was taken up by isolated myocytes in a time-dependent biphasic manner. Ru360 (10 μm) applied outside intact voltage-clamped ventricular myocytes prevented Ca2+ uptake into mitochondria in situ where the cells were progressively loaded with Ca2+ via sarcolemmal Na+/Ca2+ exchange by depolarization to +110 mV. We conclude that Ru360 specifically blocks Ca2+ uptake into mitochondria and can be used in intact cells. Ruthenium red is a well known inhibitor of Ca2+ uptake into mitochondria in vitro. However, its utility as an inhibitor of Ca2+ uptake into mitochondria in vivo or in situ in intact cells is limited because of its inhibitory effects on sarcoplasmic reticulum Ca2+ release channel and other cellular processes. We have synthesized a ruthenium derivative and found it to be an oxygen-bridged dinuclear ruthenium amine complex. It has the same chemical structure as Ru360 reported previously (Emerson, J., Clarke, M. J., Ying, W-L., and Sanadi, D. R. (1993) J. Am. Chem. Soc.115, 11799–11805). Ru360 has been shown to be a potent inhibitor of Ca2+-stimulated respiration of liver mitochondria in vitro. However, the specificity of Ru360 on Ca2+ uptake into mitochondria in vitro or in intact cells has not been determined. The present study reports in detail the potency, the effectiveness, and the mechanism of inhibition of mitochondrial Ca2+ uptake by Ru360 and its specificity in vitro in isolated mitochondria and in situ in isolated cardiac myocytes. Ru360 was more potent (IC50 = 0.184 nm) than ruthenium red (IC50 = 6.85 nm) in inhibiting Ca2+ uptake into mitochondria. 103Ru360 was found to bind to isolated mitochondria with high affinity (K d = 0.34 nm, B max = 80 fmol/mg of mitochondrial protein). The IC50 of 103Ru360 for the inhibition of Ca2+ uptake into mitochondria was also 0.2 nm, indicating that saturation of a specific binding site is responsible for the inhibition of Ca2+ uptake. Ru360, as high as 10 μm, produced no effect on sarcoplasmic reticulum Ca2+ uptake or release, sarcolemmal Na+/Ca2+ exchange, actomyosin ATPase activity, L-type Ca2+ channel current, cytosolic Ca2+ transients, or cell shortening. 103Ru360 was taken up by isolated myocytes in a time-dependent biphasic manner. Ru360 (10 μm) applied outside intact voltage-clamped ventricular myocytes prevented Ca2+ uptake into mitochondria in situ where the cells were progressively loaded with Ca2+ via sarcolemmal Na+/Ca2+ exchange by depolarization to +110 mV. We conclude that Ru360 specifically blocks Ca2+ uptake into mitochondria and can be used in intact cells. Since the discovery of Ca2+ transport into energized mitochondria about 30 years ago, numerous studies have been conducted on its kinetics and regulation in mitochondria isolated from various tissues (for review, see Gunter and Pfeiffer (1Gunter T.E. Pfeiffer D.R. Am. J. Physiol. 1990; 258: C755-C786Crossref PubMed Google Scholar)). It is now generally accepted that mitochondria act as a sink during cytosolic Ca2+ overload in diseased or damaged cells. However, the physiological role of Ca2+ transport into mitochondria in intact cells remains unresolved. In recent years, evidence has been accumulating that Ca2+ transport into mitochondria occurs in stimulated cells. For instance, it has been shown that mitochondria accumulate Ca2+ from an intracellular microdomain of high Ca2+ upon stimulation of cloned HeLa cells that express the Ca2+-sensitive photoprotein aequorin in mitochondria (2Rizzuto R. Brini M. Murgia M. Pozzan T. Science. 1993; 262: 744-747Crossref PubMed Scopus (1041) Google Scholar). Ca2+ uptake into mitochondria in situ has also been observed in stimulated pancreatic cells (3Glennon C.M. Bird G. St J. Takemura H. Thastrup O. Leslie B.A. Putney Jr., J.W. J. Biol. Chem. 1992; 267: 25568-25575Abstract Full Text PDF PubMed Google Scholar, 4Rutter G.A. Theler J-M. Murgia M. Wollheim C.B. Pozzan T. 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These studies indicate that mitochondria accumulate Ca2+ when the cytosolic free Ca2+ concentration ([Ca2+] c) increases in stimulated cells. In cardiac muscle cells, mitochondria appear to accumulate only about 1% of the [Ca2+] c during relaxation of a single twitch (12Bassani J.W.M. Bassani R.A. Bers D.M. J. Physiol. 1992; 453: 591-608Crossref PubMed Scopus (207) Google Scholar). This fraction is not enough to significantly affect [Ca2+] c and cell contractility. However, increase in mitochondrial matrix free Ca2+ concentration ([Ca2+] m) has been observed when myocytes were stimulated to increase [Ca2+] c (13Miyata H. Silverman H.S. Sollott S.J. Lakata E.G. Stern M.D. Hansford R.G. Am. J. Physiol. 1991; 261: H1123-H1134Crossref PubMed Google Scholar, 14DiLisa F. Gambassi G. Spurgeon H. Hansford R.G. Cardiovasc. Res. 1993; 27: 1840-1844Crossref PubMed Scopus (45) Google Scholar, 15Sheu S-S. Jou M-J. J. Bioenerg. 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J. 1996; 70: 2571-2580Abstract Full Text PDF PubMed Scopus (50) Google Scholar). The significance of Ca2+ uptake into mitochondria and increase in [Ca2+] m in cardiac and other cell types, however, has not been elucidated. It has been suggested that the increase in [Ca2+] m may stimulate Ca2+-sensitive matrix dehydrogenases, and thus augment the rate of ATP synthesis to meet the heightened energy demand (21Denton R.M. McCormack J.G. FEBS Lett. 1980; 119: 1-8Crossref PubMed Scopus (261) Google Scholar, 22Hansford R.G. Rev. Physiol. Biochem. Pharmacol. 1985; 102: 1-72Crossref PubMed Google Scholar, 23McCormack J.G. Halstrap A.P. Denton R.M. Physiol. Rev. 1990; 70: 391-425Crossref PubMed Scopus (1196) Google Scholar, 24Cox D.A. Matlib M.A. J. Biol. Chem. 1993; 268: 938-947Abstract Full Text PDF PubMed Google Scholar, 25Cox D.A. Matlib M.A. Trends Pharmacol. Sci. 1993; 14: 408-413Abstract Full Text PDF PubMed Scopus (100) Google Scholar, 26Wan B. LaNoue K.F. Cheung J.Y. Scaduto Jr., R.C. J. Biol. Chem. 1989; 264: 13430-13439Abstract Full Text PDF PubMed Google Scholar). Verification of this hypothesis requires demonstration of Ca2+ uptake into mitochondria and the consequent increase in matrix NAD(P)H and ATP synthesis in stimulated cardiac myocytes. The conclusions of the studies referred to above are based on presumed localization of Ca2+-sensitive dyes in mitochondria and their localization, or use of nonspecific inhibitors to block Ca2+ uptake into mitochondria. Moreover, the electron probe microanalysis approach in isolated hearts failed to detect any significant increase in mitochondrial total calcium content with isoproterenol stimulation, and yet an increase in pyruvate dehydrogenase activity was observed (27Moravec C.M. Desnoyer R.W. Milovanovic M. Schluchter M.D. Bond M. Am. J. Physiol. 1997; 273: H1432-H1439PubMed Google Scholar). A specific inhibitor of Ca2+ uptake into mitochondria will be helpful to elucidate the role of Ca2+ uptake into mitochondria in cardiac muscle and to clarify certain controversies. Ruthenium red (RR) 1The abbreviations used are: RR, ruthenium red; SR, sarcoplasmic reticulum; SL, sarcolemmal; MOPS, 3-(N-morpholino)propanesulfonic acid; BES, 2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid. has been shown to inhibit Ca2+ uptake into isolated mitochondria (28Moore C.L. Biochem. Biophys. Res. Commun. 1971; 42: 298-305Crossref PubMed Scopus (481) Google Scholar, 29Reed K.C. Bygrave F.L. Biochem. J. 1974; 140: 143-155Crossref PubMed Scopus (296) Google Scholar). However, it was also shown to inhibit Ca2+ release from SR (30Chamberlain B.K. Volpe P. Fleischer S. J. Biol. Chem. 1984; 259: 7547-7553Abstract Full Text PDF PubMed Google Scholar). It was found to produce both positive or negative inotropic effects in isolated rat hearts depending on its concentration in the perfusion solution (31Gupta M.P. Innes I.R. Dhalla N.S. Am. J. Physiol. 1988; 255: H1413-H1420PubMed Google Scholar). These effects of RR were attributed to its ability to inhibit SR Ca2+ release or sarcolemmal Na+/Ca2+ exchange. The inhibition of Ca2+ uptake into mitochondria however was shown to be due to a contaminant in the commercial preparations of RR (32Reed K.C. Bygrave F.L. FEBS Lett. 1974; 46: 109-114Crossref PubMed Scopus (27) Google Scholar). Recently, an oxygen-bridged dimeric ruthenium amine complex, which absorbs light at 360 nm (named Ru360), has been reported to inhibit Ca2+-stimulated respiration in isolated liver mitochondria (33Ying W-L. Emerson J. Clarke M.J. Sanadi D.R. Biochemistry. 1991; 30: 4949-4952Crossref PubMed Scopus (144) Google Scholar, 34Emerson J. Clarke M.J. Ying W-L. Sanadi D.R. J. Am. Chem. Soc. 1993; 115: 11799-11805Crossref Scopus (57) Google Scholar). This compound would be an extremely useful tool for elucidating the role or the contribution of Ca2+ uptake into mitochondria in vivo in isolated hearts or in situ in isolated cardiomyocytes if it penetrates the cell membrane but does not affect other cellular Ca2+ transport processes or cardiac cell contractility. To date, the specificity of Ru360 in inhibiting Ca2+ uptake into mitochondria in vitro or in intact cells has not been determined. The objectives of this study are to determine the: 1) potency, effectiveness, binding, and mechanism of action of Ru360 on Ca2+ uptake into mitochondria in vitro; 2) effects of Ru360 on processes involved in cardiac contraction, such as SR Ca2+ uptake and release, SL Na+/Ca2+ exchange, L-type Ca2+ current, and myofibrilar actomyosin ATPase; 3) effects of Ru360 on [Ca2+] c transient and cell shortening; and 4) uptake of Ru360 into intact cardiac myocytes and inhibition of Ca2+ uptake into mitochondria in situ. The results demonstrate that Ru360 binds to mitochondria with high affinity and specifically blocks Ca2+ uptake into mitochondria in vitro and in situ in intact myocytes without affecting other cellular processes involved in cardiac contraction. A procedure similar to that described by Ying et al. (33Ying W-L. Emerson J. Clarke M.J. Sanadi D.R. Biochemistry. 1991; 30: 4949-4952Crossref PubMed Scopus (144) Google Scholar) was used to synthesize Ru360 starting with RuCl3 (Sigma). The purified and crystallized preparation was reddish-brown and exhibited λmax at 360 nm in distilled water and virtually no trace of light absorbance at 533 nm, unlike RR (K & K Co./ICN product) which exhibited λmax at 533 nm and a trace of light absorbance at 360 nm. 103Ru360 was synthesized from103RuCl3 by a procedure similar to that described by Ying et al. (33Ying W-L. Emerson J. Clarke M.J. Sanadi D.R. Biochemistry. 1991; 30: 4949-4952Crossref PubMed Scopus (144) Google Scholar). The molecular structure and crystal packing were determined by x-ray crystallography. A platelike crystal (approximately 0.4 × 0.2 × 0.1 mm) was mounted onto the tip of a glass fiber with epoxy resin. Intensity data at low (233 K) and room temperature (293 K) were collected in a Siemens molecular analytical research tool (SMART, v4.05) CCD diffractometer on Mo Kα radiation (analytical x-ray instruments, Siemens, Madison, WI). The data frames were processed, and appropriate corrections for decay and Lorentz polarization effects were applied using Siemens area detector INTegration routine (SAINT, v4.05). Semiempirical absorption and beam corrections were applied using Siemens area detector ABSorption correction routine (SADABS, by G. M. Sheldrick, University of Goettengen, Germany). The structure was solved by a combination of direct method of crystal analysis (SHELXTL, v5.03, by G. M. Sheldrick) and the difference Fourier technique, and refined by full-matrix least squares on F 2. Non-hydrogen atoms were refined with anisotropic displacement parameters with the exception of C #l, which was refined isotropically. The formate atoms (O #2, C #l, O #3) were held at the atomic positions where first located during the subsequent refinement. C #l showed large thermal motion, which when left to refine, led eventually to an unstable refinement. The positions of H-atom were calculated and treated with a standard riding model. The largest residual electron density was located near the disordered formate ligand. Mitochondria were isolated from rat cardiac ventricles by the method of Matlib et al. (35Matlib M.A. Rouslin W. Vaghy P.L. Schwartz A. Methods Pharmacol. 1984; 5: 25-37Google Scholar). Male Wistar rats weighing about 300 g were anesthetized with 30 mg of nembutal/kg. The chest cavity was surgically opened, the heart excised, and immediately placed in ice-cold saline solution. After rinsing, the atria and aorta were cut off, and the ventricles immersed in an ice-cold medium containing 180 mmKCl, 10 mm EGTA, and 0.5% bovine serum albumin, pH 7.2. All subsequent steps were carried out at 0–4 °C. The tissue was weighed, minced with scissors, and homogenized with a glass-Teflon tissue homogenizer (Thomas Scientific, size C) according to procedure B (35Matlib M.A. Rouslin W. Vaghy P.L. Schwartz A. Methods Pharmacol. 1984; 5: 25-37Google Scholar). Mitochondria were isolated by differential centrifugation (35Matlib M.A. Rouslin W. Vaghy P.L. Schwartz A. Methods Pharmacol. 1984; 5: 25-37Google Scholar). The first crude mitochondrial pellet was resuspended in a medium containing 180 mm KCl and 10 mm EGTA, pH 7.4. The last wash was carried out in a medium containing 180 mmKCl and 0.05 mm EGTA, pH 7.2. The final pellet was suspended in this medium at 40–50 mg protein/ml. Protein concentration was determined by the Lowry et al. (36Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) method using bovine serum albumin to construct a linear standard curve. The rate of Ca2+ uptake into isolated mitochondria and the rate of Na+-induced Ca2+ release at 37 °C were determined spectrophotometrically using arsenazo III (37Vaghy P.L. Johnson J.D. Matlib M.A. Wang T. Schwartz A. J. Biol. Chem. 1982; 257: 6000-6002Abstract Full Text PDF PubMed Google Scholar). The assay medium (3 ml) contained 120 mm KCl, 10 mm MOPS-KOH buffer (pH 7.2), 5 mm pyruvate, 5 mm malate, 2 mm potassium phosphate buffer (pH 7.2), 50 μmarsenazo III, and 1 mg of protein. Ru360 or RR, at the desired final concentration, was added 1 min before Ca2+ uptake was initiated with the addition of 50 nmol of CaCl2. To measure the rate of Na+-induced Ca2+ release, the mitochondria were allowed to accumulate added Ca2+ (50 nmol of CaCl2) as described above. When the uptake of Ca2+ was completed, Ru360 or RR was added. One minute after the addition of Ru360 or RR, Na+-induced Ca2+ release was initiated by adding 10 mm NaCl. Isolated heart mitochondria (7 mg of protein) were incubated at 22 °C in 35 ml of medium containing 120 mmKCl, 10 mm MOPS-KOH buffer (pH 7.2), and 0.1–1 nm103Ru360. After 30 min of incubation, the samples were centrifuged at 20,000 × g for 10 min, and the supernatant was discarded. The surface of the pellet was gently rinsed twice with the above medium. The pellet was resuspended in a small volume, transferred into a test tube, and counted in a gamma counter. Nonspecific binding was determined by incubating the samples containing 0.1–1 nm103Ru360 in the presence of 10 μm unlabeled Ru360. Specific binding was determined by subtracting the nonspecific binding from the total binding. The rate of Ca2+ uptake into SRin situ in digitonin-permeabilized rat cardiomyocytes in the presence and absence of Ru360 or RR (K & K/ICN Product) was determined at 37 °C in a medium containing 100 mm NaCl, 11 mm glucose, 20 mm BES, 0.2 mm EGTA, 19 μm rotenone, 10 μm oligomycin, 1 mm dithiothreitol, 10 mm phosphocreatine, 0.2 units/ml creatine phosphokinase, 10 mm Mg-ATP, and45CaCl2 to maintain a free Ca2+ concentration of 1 μm at pH 7.2 (38Wimsatt D.K. Hohl C.M. Brierley G.P. Altschuld R.A. J. Biol. Chem. 1990; 265: 14849-14857Abstract Full Text PDF PubMed Google Scholar). The increase in the rate of Ca2+ uptake in the presence of RR was used as an indication of inhibition of Ca2+ release from SR as established previously (38Wimsatt D.K. Hohl C.M. Brierley G.P. Altschuld R.A. J. Biol. Chem. 1990; 265: 14849-14857Abstract Full Text PDF PubMed Google Scholar). SR vesicles from rat hearts were isolated according to a previously described procedure (39Kranias E.G. Schwartz A. Jungmann R.A. Biochim. Biophys. Acta. 1982; 709: 28-37Crossref PubMed Scopus (47) Google Scholar). The excised hearts were rinsed with ice-cold saline, immediately frozen in liquid nitrogen, and stored at −70 °C. The frozen hearts were powdered with stainless steel mortar and pestle in liquid nitrogen, and the powder was suspended in a solution containing 300 mm sucrose and 20 mmTris-maleate, pH 7.0. The suspension was homogenized (20 strokes at about 100 rpm) in a glass Potter Elvehjelm fitted with a Teflon piston. The homogenate was centrifuged at 1000 × g for 15 min and the supernatant was saved. The pellet was resuspended in the same solution, homogenized, and centrifuged at 1000 × g for 15 min, and the subsequent supernatant was combined with the saved supernatant. The combined supernatant was centrifuged at 10,000 ×g for 20 min and was filtered through four layers of cheesecloth. KCl was added to a final concentration of 0.6m. The solution was centrifuged at 100,000 ×g for 1 h, and the pellet was resuspended in same volume of 300 mm sucrose and 20 mm Tris-maleate buffer (pH 7.0). The suspension was centrifuged at 100,000 ×g for 1 h, and the pellet was resuspended in a small volume of 20 mm Tris-maleate, 300 mm sucrose, and 100 mm KCl, pH 7.0. The rate of Ca2+ uptake into SR vesicles was determined at 37 °C in a medium (1.5 ml) containing 40 mm imidazole, 95 mm KCl, O.5 mm EGTA, 5 mmpotassium oxalate, 5 mm MgCl2, 5 mmATP, 5 mm NaN3, 1 μm RR, and 70 μg of protein with different concentrations of free Ca2+ by using different amounts of 45CaCl2 and CaCl2 in buffered EGTA solution to yield the desired free Ca2+ concentration at pH 7.0 (40Kim H.W. Steenaart N.A. Ferguson D.G. Kranias E.G. J. Biol. Chem. 1990; 265: 1702-1709Abstract Full Text PDF PubMed Google Scholar). SL vesicles from rat heart were isolated according to a procedure described previously (41Matlib M.A. Am. J. Physiol. 1988; 255: C323-C330Crossref PubMed Google Scholar), with the exception that the supernatant solution, after the first 8000 ×g centrifugation and sedimentation of mitochondria, was used for the isolation of sarcolemmal vesicles. Ca2+ uptake into 150 mm Na+-loaded SL vesicles at 10 μm free Ca2+ was determined using45CaCl2 as a tracer and Millipore filtration technique according to a previously described procedure (41Matlib M.A. Am. J. Physiol. 1988; 255: C323-C330Crossref PubMed Google Scholar). Myofibrils from rat heart were isolated by homogenization and centrifugation according to a previously described procedure (42Savabi F. Kirsch A. J. Mol. Cell. Cardiol. 1991; 23: 1323-1333Abstract Full Text PDF PubMed Scopus (34) Google Scholar). Ca2+-stimulated actomyosin ATPase activity in the presence and absence of Ru360 was determined according to a previously described procedure (43Pagani E.D. Solaro R.J. Methods Pharmacol. 1984; 5: 49-61Google Scholar), in a solution containing 60 mm KCl, 30 mm imidazole, 7.5 mmMgCl2, 5 mm sodium ATP, 2.4 μmthapsigargin to inhibit SR Ca2+-ATPase, 5 mmNaN3 to inhibit mitochondrial ATPase, 1 mm EGTA and CaCl2 to yield 10 μm free Ca2+ at pH 7.0. Cardiac myocytes from adult rat heart were isolated as described previously (44Katsube Y. Yokoshiki H. Nguyen L. Sperelakis N. Eur. J. Pharmacol. 1996; 317: 391-400Crossref PubMed Scopus (31) Google Scholar). The isolated cells were placed into a small chamber (1.4 ml which contained the external test solution) on the stage of an inverted microscope. The cells were constantly perfused with the external test solution at rate of 1.8 ml/min. The external test solution contained in mm: 150 tetraethylammonium chloride, 1.8 CaCl2, 0.5 MgCl2, 3,4-aminopyridine, 3 Hepes, 5.5 glucose, pH 7.4 adjusted with HCl. Voltage-clamp recordings were performed in whole-cell configuration of the patch-clamp method by using patch-clamp amplifier (Axopatch-1D, Axon Instruments, Foster City, CA) and fire-polished borosilicate glass pipettes (World Precision Instruments, Sarasota, FL) with resistance of 2–6 MΩ when filled with pipette solution containing in mm: 110 CsOH, 20 CsCl, 110l-glutamic acid, 3 MgCl2, 5 disodium ATP, 5 disodium creatine phosphate, 10 EGTA, 5 Hepes, pH 7.2 adjusted with CsOH. The I Ca(L) were elicited from a holding potential of −40 mV to test potential of +10 mV for 300 ms every 15 s in cells untreated or pretreated with 10 μmRu360 at 22–25 °C. Myocytes were isolated from rat hearts as described previously (38Wimsatt D.K. Hohl C.M. Brierley G.P. Altschuld R.A. J. Biol. Chem. 1990; 265: 14849-14857Abstract Full Text PDF PubMed Google Scholar). The myocyte preparations used in this study were about 85% rod-shaped and Ca2+ tolerant. To determine the uptake of 103Ru360, 2 × 105 cells were suspended in 1 ml of Joklick's medium (Life Technologies, Inc.) containing 1 μm103Ru360 and incubated at 37 °C. At 1, 3, 5, 10, 15, 20, and 30 min after the addition of103Ru360, the cells in Microfuge tubes were sedimented by centrifugation at 12,000 × g for 15 s and washed twice with 1 ml of Joklick's medium without Ru360. The pellet was counted in a gamma counter. The amount (picomoles) of Ru360 taken up at each time point was calculated from the specific activity of the103Ru360. Myocytes from rat heart were isolated according to Wimsatt et al. (38Wimsatt D.K. Hohl C.M. Brierley G.P. Altschuld R.A. J. Biol. Chem. 1990; 265: 14849-14857Abstract Full Text PDF PubMed Google Scholar) and suspended in Krebs-Henseleit medium containing 1 mmCaCl2 and 25 mm Hepes buffer (pH 7.4). The cells were loaded with indo-1 by incubating cells in 3 μmindo-1/AM for 20 min at 37 °C. The cells were washed and further incubated at room temperature for 30 min to complete the conversion of indo-1 ester to its free acid. The cells were allowed to attach on the surface of a glass coverslip in a plastic chamber (0.3-ml volume). The cells were continuously perfused at a rate of 0.5 ml/min with buffered Krebs-Henseleit solution containing 1 mm CaCl2at 22 °C in the absence or presence of 10 μm Ru360. They were field-stimulated at 0.2 Hz (pulses of 4-ms duration) with platinum wires attached to a Grass S9 stimulator. Cell shortening was recorded with a video-edge detection system (Crescent Electronics, Salt Lake City, UT). Cytosolic indo-1 fluorescence emission ratio of 405 and 485 nm with 365 nm excitation was recorded with a P. T. I. Deltascan Photometer (Photon Technology International, Monmouth, NJ) attached to a Nikon Diaphot-200 inverted microscope. Ventricular myocytes were isolated from adult ferret hearts (45Bassani J.W.M. Bassani R.A. Bers D.M. J. Physiol. 1994; 476.2: 279-293Crossref Scopus (561) Google Scholar). The effect of Ru360 was determined under voltage-clamped conditions in which the cytosol was progressively loaded with Ca2+ via the SL Na+/Ca2+ exchanger as described previously (46Zhou Z. Matlib M.A. Bers D.M. J. Physiol. 1998; 507: 379-403Crossref PubMed Scopus (95) Google Scholar). Standard whole-cell recording techniques and Axopatch-1B amplifier (Axon Instruments) were used for electrophysiological recordings. The holding potential was −40 mV. To evoke Ca2+ influx into the cell, depolarization pulses to +110 mV were applied, which allows Ca2+ entry via the Na+/Ca2+ exchanger (46Zhou Z. Matlib M.A. Bers D.M. J. Physiol. 1998; 507: 379-403Crossref PubMed Scopus (95) Google Scholar). Signals for whole-cell current, cell contraction, and indo-1 fluorescence signals at two wavelengths (400 and 500 nm) were simultaneously recorded using pClamp 6 (Axon Instruments) and an IBM-PC compatible computer at a sampling rate of 125–500 Hz. Intracellular Ca2+ signals were detected as described previously (46Zhou Z. Matlib M.A. Bers D.M. J. Physiol. 1998; 507: 379-403Crossref PubMed Scopus (95) Google Scholar). The cells were loaded with indo-1 by incubation in Tyrode solution containing 5 μmindo-1/AM for 40–45 min at 37 °C. This condition allows loading of the cells with 0.5–1.0 mm indo-1, approximately 75% of which was found to be in mitochondria (46Zhou Z. Matlib M.A. Bers D.M. J. Physiol. 1998; 507: 379-403Crossref PubMed Scopus (95) Google Scholar). The indo-1 fluorescence was converted into intracellular free Ca2+ concentration ([Ca2+] i), representing both [Ca2+] c and [Ca2+] m, by two different methods. In the first method, [Ca2+] i was calculated from the indo-1 fluorescence ratio according to Grynkiewicz et al. (47Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar) where R min = 0.68,R max = 6.31, K d = 0.844 μm, and β = 4.35 are calibration/system constants. The [Ca2+] i was calculated based on all of the intracellular indo-1 fluorescence and represented mixed signals from cytosolic and intracellular compartments (e.g.mitochondria). [Ca2+]i=Kd·β·{(R−Rmin)/(Rmax−R)}Equation 1 In the second method, [Ca2+] i was inferred from the myocyte contraction (ΔL) measured by the video-edge-detection system (46Zhou Z. Matlib M.A. Bers D.M. J. Physiol. 1998; 507: 379-403Crossref PubMed Scopus (95) Google Scholar). The [Ca2+] i was calculated from the ΔL signal ([Ca2+] CL) given by the modified Hill equation [Ca2+]i=Kdc·[ΔL/(ΔLmax−ΔL)]1/2+0.07(μM)Equation 2 where K dc is the [Ca2+] at half-maximal contraction (0.8 μm) and ΔL max is the maximum extent of the cell shortening at very high [Ca2+] (ΔL max = 37% of resting cell length). Equation 2 assumes that the cell has its maximum length before stimulation (ΔL = 0%) and the resting [Ca2+] c = 0.07 μm. The chemical structure of the compound is presented in Fig. 1. The compound we synthesized has two ruthenium amine-formate nuclei bridged with an oxygen atom. It has three positive charges. Each ruthenium atom is positively charged with the remaining charge delocalized between the Ru-O-Ru bridge. The deduced chemical formula is C2H26N8O5Ru2Cl3and the calculated molecular weight is 550.5. A solution of 18 μm Ru360 in distilled water exhibited maximum light absorbance at 360 nm, with no detectable absorbance at 533 nm indicating that the preparation is free from ruthenium red contamination (not shown). The x-ray crystallographic data were identical to Ru360 reported previously (34E
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