ryanodine and azumolene

Mutations in the ryanodine type 1 receptor (RyR1) are causative for malignant hyperthermia. Studies in human B lymphocytes have shown that measurement of RyR1-mediated intracellular Ca(2+) (Ca(2+)(i)) release can differentiate between normal and malignant hyperthermia-susceptible individuals. The authors have further developed the B-cell assay by pharmacologically characterizing RyR1-mediated Ca release in two normal human B-cell lines and demonstrating increased sensitivity of lymphocytes to the RyR1 agonist 4-chloro-m-cresol (4-CmC) in the porcine model of MH.. Ca(2+)(i) was measured fluorometrically using fura-2 in populations of cells in suspension or with fluo-4 in single cells using confocal microscopy. The Dakiki and PP normal human B cell lines were used, as well as lymphocytes obtained from normal and malignant hyperthermia-susceptible pigs. 4-CmC was used to elicit RyR1-mediated Ca release; all experiments were performed in the absence of external Ca(2+).. EC(50) values for 4-CmC were 0.98 and 1.04 mm for Dakiki and PP cells, respectively, demonstrating reproducibility. The 4-CmC-induced increase in Ca(2+)(i) was eliminated by thapsigargin and was unaffected by xestospongin C. The Ca(2+)(i) increase was separable from mitochondrial stores and was inhibited by azumolene. Caffeine did not induce Ca(2+)(i) release, but ryanodine depleted intracellular stores by 50%. Lymphocytes from pigs carrying the Arg614Cys mutation in RyR1 showed increased sensitivity to 4-CmC (EC(50) = 0.47 vs. 0.81 mm for cells derived from normal animals).. RyR1-mediated Ca(2+) signals can be pharmacologically distinguished from other intracellular sources in human B cells, and alterations of RyR1 function can be successfully detected using Ca(2+) release from intracellular stores as an end point.

Topics: Animals; B-Lymphocytes; Caffeine; Calcium; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Cresols; Dose-Response Relationship, Drug; Humans; Imidazoles; Malignant Hyperthermia; Oxazoles; Ryanodine; Ryanodine Receptor Calcium Release Channel; Swine

Dantrolene, an intracellularly acting skeletal muscle relaxant, inhibits Ca2+ release from the sarcoplasmic reticulum during excitation-contraction coupling by an unknown mechanism. The drug is used to treat malignant hyperthermia, a genetic sensitivity to volatile anesthetics which results in the massive release of intracellular Ca2+ from affected skeletal muscle. We hypothesize that determination of the site of action of dantrolene will lead to further understanding of the regulation of sarcoplasmic reticulum calcium release. We report the identification of specific dantrolene binding sites in porcine skeletal muscle sarcoplasmic reticulum using a rapid filtration binding assay for [3H]dantrolene. The binding isotherm in the heavy sarcoplasmic reticulum fraction indicates a single binding site with a Kd of 277 +/- 25 nM and a Bmax of 13.1 +/- 1.5 pmol/mg of protein. Pharmacological specificity is characterized by inhibition of [3H]dantrolene binding with unlabeled dantrolene, or azumolene, a physiologically active congener, but not with aminodantrolene, which is physiologically inactive. Drug binding is maximal at pH 6.5-7.5, requires no Ca2+ or Mg2+, and is inhibited by salt concentrations above 100 mM. [3H]Dantrolene binding is greatest in the sarcoplasmic reticulum, which contains the ryanodine receptor, the primary calcium release channel. No binding is detected in the fractions enriched for sarcolemma or transverse tubules. We suggest that dantrolene inhibits calcium release from the sarcoplasmic reticulum by either direct or indirect interaction with the ryanodine receptor.

Topics: Animals; Binding Sites; Binding, Competitive; Calcium; Calcium Channels; Dantrolene; Hydrogen-Ion Concentration; Imidazoles; Magnesium; Membranes; Muscle Fibers, Fast-Twitch; Muscle Proteins; Osmolar Concentration; Oxazoles; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Subcellular Fractions; Swine

We tested whether the hydantoin muscle relaxants dantrolene, azumolene, or aminodantrolene could alter the binding of [3H]PN200-110 to transverse tubule dihydropyridine receptors or the binding of [3H]ryanodine to junctional sarcoplasmic reticulum Ca2+ release channels. All three drugs inhibited [3H]PN200-110 binding with azumolene (IC50 approximately 20 microM) 3-5 times more potent than dantrolene or aminodantrolene. In contrast, 100 microM azumolene and dantrolene produced a small inhibition of [3H]ryanodine binding (less than 25%) while aminodantrolene was essentially inert. Hence there was a preferential interaction of hydantoins with dihydropyridine receptors instead of ryanodine receptors. Skeletal muscle dihydropyridine receptors may participate in the mechanism of action of dantrolene and azumolene.

Topics: Animals; Caffeine; Calcium Channels; Dantrolene; Dihydropyridines; Imidazoles; Isradipine; Malignant Hyperthermia; Muscle Relaxants, Central; Muscles; Oxazoles; Receptors, Cholinergic; Receptors, Nicotinic; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum; Swine; Tritium

The mechanism of doxorubicin-induced Ca2+ release from skeletal and cardiac muscle sarcoplasmic reticulum (SR) was studied by examining the effects of azumolene (a water soluble dantrolene analog) on doxorubicin-mediated Ca2+ release and ryanodine binding. Doxorubicin induced a rapid Ca2+ release from both skeletal and cardiac SR in a similar concentration range (EC50 = 5-10 microM). Maximal doxorubicin-induced Ca2+ release was seen at 2 and 0.2 microM Ca2+ for skeletal and cardiac SR, respectively. Addition of 400 microM azumolene caused approx. 30% inhibition of doxorubicin-induced Ca2+ release from both skeletal and cardiac SR; skeletal SR had significantly higher sensitivity to azumolene than cardiac SR. In the presence of Ca2+, doxorubicin increased [3H]ryanodine binding to both skeletal and cardiac SR; whereas in the absence of Ca2+, doxorubicin led to significant ryanodine binding to skeletal SR, but not to cardiac SR. In both types of SR, doxorubicin-activated, but not Ca2+ activated ryanodine binding was inhibited by azumolene. Azumolene sensitivity for inhibition of doxorubicin-activated ryanodine binding was much higher in skeletal SR than cardiac SR, consistent with the results for effects of azumolene on Ca2+ release. Our results are consistent with the possibility that azumolene inhibits doxorubicin binding by direct competition for the drug receptor(s).

Topics: Animals; Binding, Competitive; Calcium; Doxorubicin; Imidazoles; Muscles; Myocardium; Oxazoles; Rabbits; Receptors, Cholinergic; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum