Drug-induced calcium release from heavy sarcoplasmic reticulum of skeletal muscle.

Calcium release from isolated heavy sarcoplasmic reticulum of rabbit skeletal muscle by several calmodulin antagonistic drugs was measured spectrophotometrically with arsenazo III and compared with the properties of the caffeine-induced calcium release. Trifluoperazine and W7 (about 500 microM) released all actively accumulated calcium (half-maximum release at 129 microM and 98 microM, respectively) in the presence 0.5 mM MgCl2 and 1 mg/ml sarcoplasmic reticulum protein; calmidazolium (100 microM) and compound 48/80 (70 micrograms/ml) released maximally 30-40% calcium, whilst bepridil (100 microM) and felodipin (50 microM) with calmodulin antagonistic strength similar to trifluoperazine (determined by inhibition of the calcium, calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum) did not cause a detectable calcium release, indicating that this drug-induced calcium release is not due to the calmodulin antagonistic properties of the tested drugs. Calcium release of trifluoperazine, W7 and compound 48/80 and that of caffeine was inhibited by similar concentrations of magnesium (half-inhibition 1.4-4.2 mM compared with 0.97 mM for caffeine) and ruthenium red (half-inhibition for trifluoperazine, W7 and compound 48/80 was 0.22 microM, 0.08 microM and 0.63 micrograms/ml, respectively, compared with 0.13 microM for caffeine), suggesting that this drug-induced calcium release occurs via the calcium-gated calcium channel of sarcoplasmic reticulum stimulated by caffeine or channels with similar properties.

Calcium release from calmodulin and its C-terminal or N-terminal halves in the presence of the calmodulin antagonists phenoxybenzamine and melittin measured by stopped-flow fluorescence with Quin 2 and intrinsic tyrosine. Inhibition of calmodulin-dependent protein kinase of cardiac sarcoplasmic reticulum.

Calcium dissociation from the C-terminal and N-terminal halves of calmodulin, intact bovine brain calmodulin and the respective phenoxybenzamine complexes or melittin complexes was measured directly by stopped-flow fluorescence with the calcium chelator Quin 2 and, when possible, also by protein fluorescence using endogenous tyrosine fluorescence by mixing with EGTA. Calcium dissociation from the C-terminal half of calmodulin, which contains only the two high-affinity calcium-binding sites, and from intact calmodulin was monophasic, with good correlation of the rates of calcium dissociation obtained by the two methods. The apparent rates with Quin 2 and endogenous tyrosine fluorescence were 13.4 s-1 and 12.8 s-1, respectively, in the C-terminal half and 10.5 s-1 and 10.8 s-1, respectively, in intact calmodulin (pH 7.0, 25 degrees C, 100 mM KCl). Alkylation of the C-terminal half resulted in a biphasic calcium dissociation (Quin 2: kobs 1.90 s-1 and 0.73 s-1 respectively; tyrosine: kobs 1.65 s-1 and 0.61 s-1 respectively). Alkylation of intact calmodulin resulted in a four-phase calcium dissociation measured with Quin 2 (kobs 85.3 s-1, 11.1 s-1, 1.92 s-1 and 0.59 s-1); the latter two phases are assumed to represent calcium release from high-affinity sites since they correspond to the biphasic tyrosine fluorescence change in intact alkylated calmodulin (kobs 2.04 s-1 and 0.53 s-1 respectively) and the rate parameters determined in the C-terminal half. Evidently perturbation of the calcium-binding sites by alkylation reduces the rate of calcium dissociation and allows a distinction to be made between dissociation from each of the two high-affinity sites as well as the distinct conformational change on dissociation of each calcium. Alkylation of the N-terminal half resulted in biphasic calcium release with rates (kobs 153 s-1 and 10.9 s-1 respectively) similar to those observed in intact alkylated calmodulin. The rates of calcium dissociation from calmodulin-melittin or fragment-melittin complexes, measured with Quin 2, were slower and monophasic in the C-terminal half (kobs 1.12 s-1), biphasic in the N-terminal half (kobs 140 s-1 and 26.8 s-1 respectively) and triphasic in intact calmodulin (kobs 126 s-1, 12.1 s-1 and 1.38 s-1). Calmodulin antagonists thus increase the apparent calcium affinity of high and low-affinity sites mainly due to a reduced calcium 'off rate', presumably because of conformation restrictions.(ABSTRACT TRUNCATED AT 400 WORDS)