In this report, we demonstrate the ability of the cellular thiol glutathione to modulate the ryanodine receptor from skeletal muscle sarcoplasmic reticulum. In muscle cells, cytosolic Ca 2ϩ levels are regulated by the intramuscular organelle, the sarcoplasmic reticulum (SR) 1 (1-3). Following the arrival of an action potential at the surface membrane and subsequent depolarization of the transverse tubule, the SR releases its lumenal store of Ca 2ϩ through the Ca 2ϩ release channel (CRC)/ryanodine receptor (RyR), thus triggering the contraction process. The interaction between the action potential at the transverse tubule and the release of Ca 2ϩ from the SR has been termed excitation-contraction coupling (ECC). In skeletal muscle, the molecular mechanism underlying ECC has remained unclear. Following excitation, resting Ca 2ϩ levels are reestablished through the active transport of the Ca 2ϩ back into the lumen of the SR by the Ca release by a large class of non-thiol channel stimulators, a high molecular weight complex was formed. The addition of channel inhibitors resulted in the reduction of the disulfides formed, the dissociation of key SR proteins and the exposure of hyperreactive thiols (14,15). Based on the results derived from fluorescence assays, ion flux measurements, single-channel experiments, and SDS-gel electrophoresis, the authors concluded that thiol oxidation-reduction chemistry plays a critical role in the channel gating of the SR CRC⅐RyR complex.Endogenous and exogenous redox agents have been observed to have profound effects on a wide range of ion channel systems. In addition to the SR Ca 2ϩ release channel, ion channels as varied as excitatory amino acid receptors, inositol 1,4,5-triphosphate (IP 3 )-gated Ca 2ϩ release channels, and even certain K ϩ channels have been demonstrated to be modulated by redox agents. For example, the N-methyl-D-aspartate-sensitive excitatory amino acid receptor has been shown to be modulated by both thiol oxidants and reductants (16).Glutathione is one of the most abundant low molecular weight peptides in eukaryotic cells and the most prevalent intracellular thiol. Depending on the cell type, glutathione levels have been estimated to range from 1 to 10 mM (17, 18). In the cell, glutathione acts as both a reducing agent and an antioxidant. Among its many physiological roles, glutathione * This work was supported by a grant from the American Heart Association Oregon Affiliate (to J. J. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.¶ To whom all correspondence should be addressed: Physics Dept., Portland State University, P. O. Box 751, Portland, 1 The abbreviations used are: SR, sarcoplasmic reticulum; CRC, Ca 2ϩ release channels; RyR, ryanodine receptor; RyR 1 , ryanodine receptor (skeletal isoform); ECC, excitation-contraction coupling; CPM, 7-diethylamino-3-(4Ј-maleimidylpheny...
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) = 17.2 +/- 2.4 microM; Vmax = 3.3 +/- 0.32 nmol/mg of protein/min (n = 5)]. Dihydrokainate (1 mM) inhibited uptake of radioactivity by 88 +/- 3% and had a Ki value of 65 +/- 7 microM. L-alpha-Aminoadipate (1 mM) inhibited uptake by only 25 +/- 4%. L-trans-2,4-Pyrrolidine dicarboxylate, L-serine-O-sulfate, and kainate potently inhibited transport activity with Ki values of 5.1 +/- 0.3, 56 +/- 6, and 103 +/- 9 microM, respectively (n = 3). Voltage-clamp studies of GLT1-expressing oocytes showed that, as in cortical neurons, glutamate transport was not inhibited by L-alpha-aminoadipate. Dihydrokainate was a potent inhibitor (Ki = 8 +/- 1 microM), and L-serine-O-sulfate produced a GLT1-mediated current with a K(m) value of 312 +/- 33 microM. Immunoblot analysis showed that neuronal cultures express excitatory amino acid carrier 1 (EAAC1), shown previously to be relatively insensitive to dihydrokainate, plus a trace amount of GLT1, but no GLAST. These studies establish that a major component of the glutamate transport activity of cortical neurons is dihydrokainate sensitive and distinct from the previously recognized neuronal transporter excitatory amino acid carrier 1.
Favero, Terence G., Anthony C. Zable, David Colter, and Jonathan J. Abramson. Lactate inhibits Ca2+-activated Ca2+-channel activity from skeletal muscle sarcoplasmic reticulum. J. Appl. Physiol. 82(2): 447–452, 1997.—Sarcoplasmic reticulum (SR) Ca2+-release channel function is modified by ligands that are generated during about of exercise. We have examined the effects of lactate on Ca2+- and caffeine-stimulated Ca2+ release, [3H]ryanodine binding, and single Ca2+-release channel activity of SR isolated from rabbit white skeletal muscle. Lactate, at concentrations from 10 to 30 mM, inhibited Ca2+- and caffeine-stimulated [3H]ryanodine binding to and inhibited Ca2+- and caffeine-stimulated Ca2+ release from SR vesicles. Lactate also inhibited caffeine activation of single-channel activity in bilayer reconstitution experiments. These findings suggest that intense muscle activity, which generates high concentrations of lactate, will disrupt excitation-contraction coupling. This may lead to decreases in Ca2+ transients promoting a decline in tension development and contribute to muscle fatigue.
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 (7.1-6.5) inhibited SR Ca2+ release and [3H]-ryanodine binding. In addition, increasing lactate concentrations from 2 to 20 mM inhibited [3H]ryanodine binding to SR vesicles, inhibited SR Ca2+ release, and decreased the single-channel open probability. These findings suggest that intracellular modifications that disrupt excitation-contraction coupling and decrease Ca2+ transients will promote a decline in tension development and contribute to muscle fatigue. In addition, we show that hydrogen peroxide induces Ca2+ release and increases [3H]ryanodine binding to its receptor, suggesting that reactive oxygen species produced during exercise may compromise muscle function by altering the normal gating of the SR Ca2+ release channel.
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