Chloride-induced release of actively loaded calcium from light and heavy sarcoplasmic reticulum vesicles

Summary

Light and heavy sarcoplasmic reticulum vesicles (LSR, HSR) isolated from rabbit leg muscle have been used in a study of chloride-induced Ca²⁺ release. The biochemical and morphological data indicate that LSR is derived from the longitudinal reticulum and HSR is derived from the terminal cisternae of the sarcoplasmic reticulum. LSR and HSR were both able to accumulate Ca²⁺ in the presence of ATP to amounts greater than 100 nmol Ca²⁺/mg of protein in less than 1 min. LSR and HSR each had a biphasic time course of Ca²⁺ uptake. The initial uptake was followed by a rapid release, after approximately 1 min, of 30–40% of the accumulated Ca²⁺, which was then followed by a slower phase of Ca²⁺ accumulation. Ca²⁺ taken up by the SR vesicles could be released from both the LSR and HSR by changing the anion outside the vesicles from methanesulfonate to chloride. Due to the difference in permeability between methanesulfonate and chloride, this change should result in a decreased positivity inside the vesicles with respect to the exterior. It could also result in osmotic swelling of the vesicles. Changing the ionic medium from chloride to methanesulfonate caused no release of Ca²⁺. The amount of accumulated Ca²⁺ released in 6 sec by changing the anion outside the vesicles from methanesulfonate to chloride was 30–35 nmol/mg membrane protein for LSR and HSR, respectively. Osmotic buffering with 200mm sucrose caused a slight inhibition of chloride-induced Ca²⁺ release from HSR (17%→15%) but it greatly reduced the release of Ca²⁺ from LSR (32%→15%). The specificity of Ca²⁺ release was measured using SR vesicles which were passively loaded with 10mm ²²Na⁺. LSR released five times more²²Na⁺ than HSR under same conditions as chloride-induced Ca²⁺ release occurred. Na dantrolene (20 μm) had no effect on the release of Ca²⁺ from LSR but it inhibited the chloride-induced Ca²⁺ release from HSR by more than 50%. Na dantrolene also increased the Ca²⁺ uptake in the HSR by 20% while not affecting LSR Ca²⁺ uptake. Our results indicate the presence of a chloride-induced, Na dantrolene inhibited, Ca²⁺ release from HSR, which is not due to osmotic swelling.