High affinity glutamate transport in rat cortical neurons in culture.

We assayed glutamate transport activity in cultures of rat cortical neurons containing < 0.2% astrocytes. Using [3H]L-glutamate as the tracer, sodium-dependent high affinity glutamate transport was demonstrated [K(m) = 17.

Glutathione modulates ryanodine receptor from skeletal muscle sarcoplasmic reticulum. Evidence for redox regulation of the Ca2+ release mechanism.

In this report, we demonstrate the ability of the cellular thiol glutathione to modulate the ryanodine receptor from skeletal muscle sarcoplasmic reticulum. Reduced glutathione (GSH) inhibited Ca2+-stimulated [3H]ryanodine binding to the sarcoplasmic reticulum and inhibited the single-channel gating activity of the reconstituted Ca2+ release channel. The effects of GSH on both the [3H]ryanodine binding and single-channel measurements were dose-dependent, exhibiting an IC50 of approximately 2.

Lactate inhibits Ca(2+) -activated Ca(2+)-channel activity from skeletal muscle sarcoplasmic reticulum.

Sarcoplasmic reticulum (SR) Ca(2+)-release channel function is modified by ligands that are generated during about of exercise. We have examined the effects of lactate on Ca(2+)- and caffeine-stimulated Ca2+ release, [3H]ryanodine binding, and single Ca(2+)-release channel activity of SR isolated from rabbit white skeletal muscle. Lactate, at concentrations from 10 to 30 mM, inhibited Ca(2+)- and caffeine-stimulated nodine binding to and inhibited Ca(2+)- and caffeine-stimulated [3H]ryanodine binding to and inhibited Ca(2+)- and caffeine-stimulated Ca2+ release from SR vesicles.

Thimerosal interacts with the Ca2+ release channel ryanodine receptor from skeletal muscle sarcoplasmic reticulum.

The thiol-oxidizing reagent, thimerosal, has been shown to increase the intracellular Ca2+ concentration, to induce Ca2+ spikes in several cell types, and to increase the sensitivity of intracellular Ca2+ stores to inositol 1,4,5-trisphosphate. Ryanodine-sensitive stores have also been implicated in the generation of Ca2+ oscillations induced by the addition of thimerosal. Here we report that micromolar concentrations of thimerosal stimulate Ca2+ release from skeletal muscle sarcoplasmic reticulum vesicles, inhibit high affinity [3H]ryanodine binding, and modify the channel activity of the reconstituted Ca2+ release protein.

Hydrogen peroxide stimulates the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.

Hydrogen peroxide (H2O2) at millimolar concentrations induces Ca2+ release from actively loaded sarcoplasmic reticulum vesicles and induces biphasic [3H]ryanodine binding behavior. High affinity [3H]ryanodine binding is enhanced at concentrations from 100 microM to 10 mM (3-4 fold). At H2O2 concentrations greater than 10 mM, equilibrium binding is inhibited.

Metabolic end products inhibit sarcoplasmic reticulum Ca2+ release and [3H]ryanodine binding.

Sarcoplasmic reticulum (SR) Ca2+ release channel function is modified by ligands (Mg2+, Ca2+, ATP, and H+) that are generated during a bout of exercise. We have examined the effects of changing intracellular metabolites on Ca2+ release, [3H]ryanodine binding, and single-Ca2+ release channel activity of SR isolated from white rabbit skeletal muscle. Increasing Mg2+ (from 0 to 4 mM) and decreasing pH (7.

Ca2+ binding sites of the ryanodine receptor/Ca2+ release channel of sarcoplasmic reticulum. Low affinity binding site(s) as probed by terbium fluorescence.

Fluorescence spectroscopy has been used to study the interaction of Tb3+ (as a Ca2+ analog) with the purified ryanodine receptor (RyR)/Ca2+ release channel of skeletal muscle sarcoplasmic reticulum. Tb3+ replaces Ca2+ in both the high- and the low-affinity sites. Occupation of the low-affinity site (inhibitory), but not of the high-affinity Ca2+ binding site (activating), by Tb3+ results in a strong enhanced green fluorescence (at 543 nm) and in an inhibition of ryanodine binding.

Direct evidence for the existence and functional role of hyperreactive sulfhydryls on the ryanodine receptor-triadin complex selectively labeled by the coumarin maleimide 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin.

The fluorogenic sulfhydryl probe 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) (1-50 nM) is used to characterize the functional role and location of highly reactive thiol groups on the ryanodine-sensitive Ca2+ release channel complex [i.e., ryanodine receptors (RyRs)] of skeletal and cardiac junctional sarcoplasmic reticulum (SR).