Figure 4. Ca²⁺ release from heavy SR vesicles induced by HCa.

(A) Absorbance measured in the absence of SR vesicles and in response to sequential Ca²⁺ additions in the medium or to 40 nM HCa addition. The data indicate the lack of Ca²⁺ in the purified material. (B) Heavy SR vesicles were actively loaded with Ca²⁺ by four sequential additions of 20 μM CaCl₂ in the monitoring chamber. The absorbance was monitored to show that the added Ca²⁺ was taken up by the SR vesicles. The trace relaxed close to its original baseline with CaCl₂ additions constituting approx. 70–80% of the SR loading capacity. These Ca²⁺ additions were used to calibrate the Ca²⁺ release. Addition of 125 nM HCa produces a long-lasting Ca²⁺ release from SR vesicles. Ca²⁺ (20 μM) was added along with HCa to control the extravesicular concentration of Ca²⁺. Also, external Ca²⁺ appears to act as a cofactor to the HCa effect. Residual Ca²⁺ from SR vesicles is released by the addition of 1 μM ionophore A23187. Further addition of 0.5 mM EGTA buffers the released and the basal Ca²⁺ from the system. (C) Loading of heavy SR vesicles with Ca²⁺ by six sequential additions of 20 μM CaCl₂ in the monitoring absorbance chamber. The fifth application did not produce a similar calcium release to the one observed by the co-application of 20 μM Ca²⁺ and 125 nM HCa in (B). (D) Loading of heavy SR vesicles with Ca²⁺ by four sequential additions of 20 μM CaCl₂ and a fifth one at 40 μM in the monitoring absorbance chamber. The fifth application produces a slower loading of the vesicles, but no sustained response.