Abstract
Spontaneous calcium release from purified light sarcoplasmic reticulum has been previously described (Palade, P., Mitchell, R. D., and Fleischer, S. (1983) J. Biol. Chem. 258, 8098-8107) and found to be distinct from several other forms of Ca2+ release. Ca2+ release occurs after a lag period following active Ca2+ preloading and depletion of extravesicular Ca2+. In the present study, we find that local anesthetics inhibit spontaneous Ca2+ release, in a time-dependent manner, varying considerably in the preincubation time required to exert maximal effect. At pH 7.0, hydrophilic and mostly charged local anesthetics, such as procaine, procainamide, and N-(2,6-dimethylphenyl carbamoyl methyl)triethyl ammonium bromide, inhibit Ca2+ release only after long preincubations (hours), whereas more hydrophobic local anesthetics are effective after only a short incubation (minutes) with sarcoplasmic reticulum. The more hydrophobic anesthetics take somewhat longer to reach equilibrium, as studied by inhibition of unidirectional Ca2+ efflux, and there is a direct relationship between hydrophobic partition coefficient and half-time to reach equilibrium. Agents known to inhibit permeability pathways for monovalent cations i.e. K+ channel blockers (decamethonium and n-dodecane-1, 12-N,N,N,N',N',N'-hexamethyl-bis-ammonium) or the anion blocker (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), do not inhibit spontaneous Ca2+ release. Carbonyl cyanide m-fluorophenylhydrazone, a protonophore, and gramicidin D, a monovalent cation ionophore, have no effect on Ca2+ release whether local anesthetics are present or not, while the Ca2+ ionophore A23187 relieves inhibition of Ca2+ release by local anesthetics. Ruthenium red does not inhibit spontaneous Ca2+ release. These findings suggest that the binding site(s) for local anesthetics is located on the inner face of the sarcoplasmic reticulum membrane and that local anesthetics interact directly with a Ca2+ channel rather than with other permeability pathways which might indirectly influence Ca2+ channel gating.
Highlights
Spontaneous calciumrelease from purified lighstar- esthetics interact directly with a Caz+channel rather coplasmic reticulum hasbeen previously lade, p., Mitchell, R
Local anestheticshave beenused for more than half a study, we find that local anesthetics inhibit sponta- century [1]to study anindfluence skeletal muscle contraction
Carbonyl cyanide m-fluoro- As reported recently [43]; spontaneous Ca2+ release3 rephenylhydrazone, a protonophore, and gramicidin D, quires a critical extent of calcium preloading in thepresence a monovalent cation ionophore, have no effeoctn Ca2+ of high phosphate levels and takes place, after a lag period, release whether local anesthetics are present or not, when the free [Ca], has been reduced to the submicromolar while the Ca2+ionophore A23187 relieves inhibition range
Summary
28) and enhance [5, 11,12, 19, 29,30,31,32,33,34,35,36,37,38,39,40] Ca2+ release fromSR.’ Reasons for such divergent results include variations in (a) methy1)triethyl ammonium bromide, inhibit Ca2+re- the chemical structure [5] of the anesthetics; ( b )the concenlease only after long preincubations (hours), whereas tration used [10, 11, 19, 34, 39]; (c) the assay conditions Ruthenium red of local anesthetics on spontaneouCs a2+ releasefrom skeletal does not inhibit spontaneousCa2+release. Preparation of Isolated SR and Preincubation with Local Anesthetics-Light SR, derived mostly from longitudinal sarcoplasmic reticulum of rabbit fast-twitch skeletal muscle, was isolated and purified as previously described [46]. When ethanolic solutions of drugs were used, equal amounts of alcohol were added to the control (0.5-1%)
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