Dantrolene and A13187 ionophore: specific action on calcium channels revealed by the aequorin method.
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Biomedical subjects
Publications and source records attributed to K Hainaut.
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1. Ca movements in resting and in activated single giant muscle fibres of the barnacle were analysed before and after exposure to Dantrolene Na, a synthetic hydantoin derivative. 2. In fibres micro-injected with the photoprotein aequorin, the resting rate of light emission (resting glow) reversibly decreased upon exposure to Dantrolene. Similar results were obtained if the fibre had first been equilibrated in a O Ca-1 mM-EGTA medium. 3. The influx of 45Ca into resting muscle fibres was not modified by 35 micronM Dantrolene which also failed to significantly reduce the influx of 45Ca into muscle fibres which had been depolarized by exposure to external solutions in which K+ had been increased to 60 or 200 mM. 4. In fibres micro-injected with 45Ca, the calcium efflux was reversibly decreased by Dantrolene. This effect was still observed in O Ca medium and in O Ca-ONa medium. A possible effect of Dantrolene on the Na-Ca exchange process at the outer membrane was excluded by showing that when the direction of the Ca2+ movement was inverted in aequorin-loaded fibres by the sudden removal of Na+ from the external medium, a marked increase in the resting glow was recorded which was not affected by exposure to Dantrolene. 5. It is argued that the reduction of Ca2+ efflux by Dantrolene does not result from any direct inhibitory effect on the metabolically driven Ca pump at the outer membrane, but that it is rather related to the reduction of the concentration of myoplasmic Ca2+ which is indeed demonstrated by the reduced resting glow. This in turn is thought to result from a shift in the balance between Ca2+ movements into and out of the intracellular storage sites, and namely the sarcoplasmic reticulum (SR). 6. The Ca2+ transient in aequorin-loaded fibres and the force of the isometric contraction elicited by imposed membrane depolarizations were markedly reduced by Dantrolene. The electrochemical threshold for eliciting intracellular Ca2+ release was not significantly modified. The linear relation between membrane depolarization and Ca2+ transient became less steep. The process of sequestration of myoplasmic Ca2+ back into SR was not significantly affected by Dantrolene which appeared to inhibit rather selectively the Ca2+ release from SR into the cytosol.
Dantrolene sodium, a hydantoin derivative with skeletal muscle relaxant properties, quickly reduces the cytosolic calcium concentration of the Barnacle (Balanus nubilus) giant muscle fibre, by direct action on the sercoplasmic reticulum. This inhibitory effect subsequently reduces the 45Ca efflux out of the cell although the 45Ca influx remains unchanged.
1. Ca movements were studied in single giant muscle fibres of the barnacle before and after exposure to the Ca ionophore A23187. 2. In fibres micro-injected with the photoprotein aequorin, the resting rate of light emission (resting glow) reversibly increased when the external Ca was augmented in the presence of A23187. This resulted from an increased Ca influx through the outer membrane. In zero external Ca, A23187 induced a delayed increase of the resting glow which was related to Ca leakage from intracellular storage sites. 3. When the experiment was carried out at 5 degrees C instead of 22 degrees C, the resting glow about doubled (temperature-sensitive mechanisms for maintaining a low myoplasmic Ca2+ were thus depressed) but A23187 was much less effective in increasing Ca influx in high external Ca. 4. In 45Ca efflux experiments, A23187 increased both the Ca-Ca exchange diffusion and the net Ca outflux at the outer membrane of the fibre. The Na-Ca exchange process was not affected to a significant extent. 5. The Ca2+ transient and the force of the contraction elicited by imposed membrane depolarizations (below the threshold of the propagated action potential) were markedly increased by A23187, but the electromechanical threshold was not significantly modified. The linear relation between membrane depolarizations and the area of the Ca transient became much steeper in the presence of A23187. However the relation between the area of the Ca transient and the force output was not affected which suggests that A23187 specifically involved the Ca2+ intracellular release, but not the subsequent Ca-troponin reaction. 6. A23187 potentiates by ionophoretic action the Ca movements at the outer membrane, and it also acts inside the intact muscle fibre to intensify the intracellular release of Ca.
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