Inositol 1,4,5-trisphosphate-induced Ca2+ release from the sarcoplasmic reticulum and contraction in crustacean muscle

Abstract

Intracellular applications of a fixed amount (0.2 to 8 nmol) of inositol 1,4,5-trisphosphate (InsP3) over a brief period (2 s) into barnacle muscle fibers induced vigorous contractures. Peak tension attained during the first application depended on [InsP3]; the maximum tension evoked by the injection of 8 nmol was 1.6 kg/cm2. Peak tension during a second application of a high dose of InsP3 (> 10 μM) was always smaller than that during the first application. Extracellular Ca2+ could be omitted with no measurable effects on either the amplitude or time course of the contractures evoked by InsP3. Aequorin was used to measure InsP3-evoked Ca2+ release from intracellular stores in minced muscle fibers from lobster and in skinned muscle fibers from barnacle. Provided the sarcoplasmic reticulum was preloaded with Ca2+, application of InsP3 induced a transient Ca2+ release that was [InsP3] dependent. During each transient, [Ca2+] rose rapidly to a peak value (t1/2 < 5 s) and then slowly returned (t1/2 < 100 s) to a basal level. Maximum Ca2+ release was obtained at [InsP3] < 100 μM and amounted to 4 nmol Ca2+/g of muscle, enough to increase [Ca2+]i from 0.1 to 8 μM had the Ca2+ release occurred in the intact fiber. Successive applications of a fixed amount of InsP3 elicited successive transient increases in Ca2+. The effects of [Ca2+] on the incorporation of [3H]inositol into the pools of phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate pools were measured. At 1 μM [Ca2+] the levels of the labelled phosphoinositides increased from 0.98, 0.40, and 0.17 pmol/μmol phosphorus (measured at 0.1 μM) to 1.63, 0.54, and 0.46 pmol/μmol lipid phosphorus, for phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-biphosphate, respectively. These data support the hypothesis that InsP3 could be an intracellular messenger, presumably between the site of Ca2+ entry at the level of the transverse tubular membrane and the sarcoplasmic reticulum membrane, which causes Ca2+ release from Ca2+-loaded sarcoplasmic reticulum in crustacean muscle.