RUTHENIUM RED AND COMPOUND-48/80 INHIBIT THE SMOOTH-MUSCLE PLASMA-MEMBRANE CA-2+ PUMP VIA INTERACTION WITH ASSOCIATED POLYPHOSPHOINOSITIDES

We will demonstrate that compound 48 80 and ruthenium red inhibit the smooth-muscle plasma-membrane Ca2+ pump by counteracting the stimulant effect of negatively charged phospholipids. Both substances did not affect the purified enzyme re-activated by pure phosphatidylcholine or phosphatidylinositol and measured in the absence of calmodulin, indicating that under these conditions they did not have a direct effect on the ATPase protein. Ruthenium red and compound 48 80 however inhibited the (Ca2+ + Mg2+)-ATPase in the presence of phosphatidylinositol 4-phosphate and especially phosphatidylinositol 4,5-biphosphate. The K0.5 for inhibition was 25 μM ruthenium red and 9 μg/ml of compound 48 80. The inhibition by ruthenium red developed slowly with half maximal inhibition occurring after about 75 s while that by compound 48 80 developed immediately within the time required for mixing. The efficacy of ruthenium red increased as the concentration of the acidic phospholipid increased, while no such cooperativity was observed for compound 48 80. Ruthenium red reduced the Vmax for Ca2+ without affecting the affinity for Ca2+, while compound 48 80 decreased both parameters. In conclusion, although ruthenium red and compound 48 80 affect the ATPase differently, both substances most likely inhibit the plasma-membrane Ca2+ pump by counteracting the stimulation by negatively charged phospholipids. © 1990.