DHPR activation underlies SR Ca²⁺ release induced by osmotic stress in isolated rat skeletal muscle fibers

Changes in skeletal muscle volume induce localized sarcoplasmic reticulum (SR) Ca2+ release (LCR) events, which are sustained for many minutes, suggesting a possible signaling role in plasticity or pathology. However, the mechanism by which cell volume influences SR Ca2+ release is uncertain. In the present study, rat flexor digitorum brevis fibers were superfused with isoosmotic Tyrode's solution before exposure to either hyperosmotic (404 mOsm) or hypoosmotic (254 mOsm) solutions, and the effects on cell volume, membrane potential (Em), and intracellular Ca2+ ([Ca2+]i) were determined. To allow comparison with previous studies, solutions were made hyperosmotic by the addition of sugars or divalent cations, or they were made hypoosmotic by reducing [NaCl]o. All hyperosmotic solutions induced a sustained decrease in cell volume, which was accompanied by membrane depolarization (by 14–18 mV; n = 40) and SR Ca2+ release. However, sugar solutions caused a global increase in [Ca2+]i, whereas solutions made hyperosmotic by the addition of divalent cations only induced LCR. Decreasing osmolarity induced an increase in cell volume and a negative shift in Em (by 15.04 ± 1.85 mV; n = 8), whereas [Ca2+]i was unaffected. However, on return to the isoosmotic solution, restoration of cell volume and Em was associated with LCR. Both global and localized SR Ca2+ release were abolished by the dihydropyridine receptor inhibitor nifedipine by sustained depolarization of the sarcolemmal or by the addition of the ryanodine receptor 1 inhibitor tetracaine. Inhibitors of the Na-K-2Cl (NKCC) cotransporter markedly inhibited the depolarization associated with hyperosmotic shrinkage and the associated SR Ca2+ release. These findings suggest (1) that the depolarization that accompanies a decrease in cell volume is the primary event leading to SR Ca2+ release, and (2) that volume-dependent regulation of the NKCC cotransporter contributes to the observed changes in Em. The differing effects of the osmotic agents can be explained by the screening of fixed charges by divalent ions.