Pharmacological distinction between dantrolene and ryanodine binding sites: evidence from normal and malignant hyperthermia-susceptible porcine skeletal muscle.
Dantrolene inhibits and ryanodine stimulates calcium release from skeletal-muscle sarcoplasmic reticulum (SR), the former by an unknown mechanism, and the latter by activating the ryanodine receptor (RyR), the primary Ca2+-release channel of SR. Dantrolene is used to treat malignant hyperthermia (MH), a genetic predisposition to excessive intracellular Ca2+ release upon exposure to volatile anaesthetics. Porcine MH results from a point mutation in the SR RyR that alters the open probability of the channel, and is reflected in altered [3H]ryanodine binding parameters. Specific binding sites for [3H]dantrolene and [3H]ryanodine co-distribute on SR that has been isolated by discontinuous sucrose gradient centrifugation. If the two drug-binding sites are functionally linked, [3H]dantrolene binding might be affected both by pharmacological and by genetic modulators of the functional state of the RyR. Accordingly, we compared the characteristics of [3H]dantrolene binding to porcine malignant-hyperthermia-susceptible and normal-skeletal-muscle SR, and examined the effects of RyR modulators on [3H]dantrolene binding to these membranes. Additionally, the feasibility of separating the SR binding sites for [3H]dantrolene and [3H]ryanodine was investigated. No significant differences in [3H]dantrolene binding characteristics to SR membranes from the two muscle types were detected, and the Bmax ratio for [3H]dantrolene/[3H]ryanodine was 1.4(+/-0.1):1 in both muscle types. [3H]Dantrolene binding is unaffected by the RyR modulators caffeine, ryanodine, Ruthenium Red and calmodulin, and neither dantrolene nor azumolene have any effect on [3H]ryanodine binding. Additionally, distinct peaks of [3H]dantrolene and [3H]ryanodine binding are detected in SR membranes fractionated by linear sucrose centrifugation, although no differences in protein patterns are detected by SDS/PAGE or Western-blot analysis. We suggest that the binding sites for these two drugs are pharmacologically distinct, and may exist on separate molecules.
Palnitkar
S S
SS
Department of Anesthesia, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, New Brunswick, NJ 08901, USA.
Mickelson
J R
JR
Louis
C F
CF
Parness
J
J
D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D003620:Dantrolene / Q000378:metabolism / Q000494:pharmacology*; D008305:Malignant Hyperthermia / Q000378:metabolism* / Q000473:pathology; D009125:Muscle Relaxants, Central / Q000378:metabolism / Q000494:pharmacology*; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000473:pathology; D009994:Osmolar Concentration; D011869:Radioligand Assay; D012433:Ryanodine / Q000378:metabolism / Q000494:pharmacology*; D013552:Swine
D009125:Muscle Relaxants, Central; D012433:Ryanodine; D003620:Dantrolene