OXYGEN-BRIDGED DINUCLEAR RUTHENIUM AMINE COMPLEX SPECIFICALLY INHIBITS CA2-VITRO AND IN-SITU IN SINGLE CARDIAC MYOCYTES( UPTAKE INTO MITOCHONDRIA IN)

Ruthenium red is a well known inhibitor of Ca2+ uptake into mitochondr ia 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 derivati ve and found it to be an oxygen-bridged dinuclear ruthenium amine comp lex. 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. Sec. 115, 11799-11805). Ru360 has been shown to be a poten t inhibitor of Ca2+-stimulated respiration of liver mitochondria in vi tro. However, the specificity of Ru360 on Ca2+ uptake into mitochondri a in vitro or in intact cells has not been determined. The present stu dy reports in detail the potency, the effectiveness, and the mechanism of inhibition of mitochondrial Ca2+ uptake by Ru360 and its specifici ty in vitro in isolated mitochondria and in situ in isolated cardiac m yocytes, Ru360 was more potent (IC50 = 0.184 nM) than ruthenium red (I C50 = 6.85 nM) in inhibiting Ca2+ uptake into mitochondria, (103)Ru360 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 ( 103)Ru360 for the inhibition of Ca2+ uptake into mitochondria was also 0.2 nM, indicating that saturation of a specific binding site is resp onsible for the inhibition of Ca2+ uptake. Ru360, as high as 10 mu M,p roduced no effect on sarcoplasmic reticulum Ca2+ uptake or release, sa rcolemmal Na+/Ca2+ exchange, actomyosin ATPase activity, L-type Ca2+ c hannel current, cytosolic Ca2+ transients, or cell shortening. (103)Ru 360 was taken up by isolated myocytes in a time-dependent biphasic man ner. Ru360 (10 mu M) applied outside intact voltage-clamped ventricula r myocytes prevented Ca2+ uptake into mitochondria in situ where the c ells were progressively loaded with Ca2+ via sarcolemmal Na+/Ca2+ exch ange by depolarization to +110 mV. We conclude that Ru360 specifically blocks Ca2+ uptake into mitochondria and can be used in intact cells.