Fig. 1.

CGP inhibits SR Ca²⁺ uptake and SERCA-mediated ATPase activity. SR microsomes were incubated in phosphate buffer containing ATP/Mg with 2 μl of CGP in DMSO (final CGP levels from 0.625 to 20 μM) or with 2 μl of DMSO (control). SR Ca²⁺ loading was started by increasing Ca²⁺ in the cuvette to 40 micromolar. A, example of Ca²⁺ uptake by porcine cardiac SR microsomes measured under control conditions and in the presence of various doses of CGP (0.625–20 μM). B, percentage of inhibition of the rate of SR Ca²⁺ loading by porcine cardiac SR microsomes versus CGP concentrations. Experimental data as in A were fitted by a single exponential function from which the initial rate of Ca²⁺ uptake was derived (see Materials and Methods). The average rate of uptake decreased by 60.2 ± 4.6% at 20 μM CGP (n = 3). From the data in A, a half-maximal inhibitory concentration of 9.9 ± 2.0 μM was estimated. C, example of CGP-induced inhibition of Ca²⁺ uptake by rabbit skeletal muscle TC microsomes. D, the drug decreased the rate of uptake by skeletal TC microsomes by 62.7 ± 7.0% with 20 μM CGP, IC₅₀ = 6.6 ± 1.2 μM (n = 4). E, decrease in NADH absorption versus time (indicative of ATPase activity) by skeletal LT microsomes enriched in SERCA. As shown, CGP (10 μM) partially inhibited the decrease of NADH levels, whereas CPZ (20 μM) completely stopped the reaction. F, CGP-induced inhibition of ATPase activity of skeletal LT microsomes incubated with free [Ca²⁺] of either 50 or 300 nM. The inhibitory action at the two free [Ca²⁺] was comparable, 31.3 ± 5.3 and 42.7 ± 6.4% respectively (n = 4).