4, 6‐Dibromo‐3‐hydroxycarbazole (an analogue of caffeine‐like Ca2+ releaser), a novel type of inhibitor of Ca2+‐induced Ca2+ release in skeletal muscle sarcoplasmic reticulum

Abstract

1. 4,6-Dibromo-3-hydroxycarbazole (DBHC) was synthesized as an analogue of bromoeudistomin D (BED), a powerful Ca2+ releaser, and its pharmacological properties were examined. 2. In Ca2+ electrode experiments, DBHC (100 microM) markedly inhibited Ca2+ release from the heavy fraction of sarcoplasmic reticulum (HSR) induced by caffeine (1 mM) and BED (10 microM). 3. DBHC (0.1 to 100 microM) inhibited 45Ca2+ release induced by Ca2+ from HSR in a concentration-dependent manner. 4. DBHC (100 microM) abolished 45Ca2+ release induced by caffeine (1 mM) and BED (10 microM) in HSR. 5. Inhibitory effects of calcium-induced calcium release (CICR) blockers such as procaine, ruthenium red and Mg2+ on 45Ca2+ release were clearly observed at Ca2+ concentrations from pCa 7 to pCa 5.5, and were decreased at Ca2+ concentrations higher than pCa 5.5 or lower than pCa 7. However, DBHC decreased Ca2+ release induced by Ca2+ over the wide range of extravesicular Ca2+ concentrations. 6. [3H]-ryanodine binding to HSR was suppressed by ruthenium red, Mg2+ and procaine, but was not affected by DBHC up to 100 microM. 7. [3H]-ryanodine binding to HSR was enhanced by caffeine and BED. DBHC antagonized the enhancement in a concentration-dependent manner. 8. 9-[3H]-Methyl-7-bromo-eudistomin D, an 3H-labelled analogue of BED, specifically bound to HSR. Both DBHC and caffeine increased the KD value without affecting the Bmax value, indicating a competitive mode of inhibition. 9. These results suggest that DBHC binds to the caffeine binding site to block Ca2+ release from HSR. This drug is a novel type of inhibitor for the CICR channels in SR and may provide a useful tool for clarifying the Ca2+ releasing mechanisms in SR.