ryanodine and ferric-chloride

The present study evaluates the effect of free radicals generated by xanthine oxidase-catalyzed oxidation of hypoxanthine on cellular function of isolated rat pancreatic acinar cells. The results show that a rapid and sustained increase in intracellular Ca2+ concentration ([Ca2+]i) preceded all other morphological and functional alterations investigated. Radical-induced [Ca2+]i increase was largely inhibited by 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester, which prevents Ca2+ release from intracellular stores, but not by Ca2(+)-depleted medium. Radicals released Ca2+ from thapsigargin-insensitive, ryanodine-sensitive intracellular stores, whereas the secretagogue caerulein at physiological concentrations mainly released Ca2+ from thapsigargin-sensitive stores. In contrast to effects of the secretagogue, radical-induced Ca2+ changes did not cause luminal protein secretion but cell death. In single-cell measurements, both secretagogue and radicals induced oscillations of [Ca2+]i. Radical-induced oscillations had a lower frequency but similar amplitude when compared with caerulein-induced oscillations. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and ryanodine, which prevented the radical-induced Ca2+ increase without altering the generation of radicals, markedly reduced the radical-induced cell damage. These results suggest that the Ca2+ increase mediates the radical-induced cell injury. The studies also indicate that not only the extent and duration but also the origin of [Ca2+]i release as well as the frequency of Ca2+ oscillations may determine whether a pancreatic acinar cell will secrete or die.

Topics: Adenosine Triphosphate; Amylases; Animals; Calcimycin; Calcium; Cells, Cultured; Ceruletide; Chlorides; Cytosol; Edetic Acid; Ferric Compounds; Free Radicals; Hypoxanthine; Kinetics; L-Lactate Dehydrogenase; Male; Oxidative Stress; Pancreas; Rats; Rats, Wistar; Ryanodine; Thapsigargin; Xanthine Oxidase

Iron is examined for its ability to modify Ca2+ transport across sarcoplasmic reticulum (SR) and to alter the binding of [3H]ryanodine to its high-affinity site on the Ca2+ release channel complex of SR preparations from rat heart. Iron(III) (added as ferric chloride) has negligible activity on active Ca2+ accumulation into SR and on the binding of [3H]ryanodine. In contrast, Fe(II) (added as ferrous sulfate) is a potent inhibitor of both Ca(2+)-induced Ca2+ release (IC50 of 29 microM) and DXR-induced Ca2+ release (IC50 of 14 microM). Iron(II) enhances the rate of active Ca2+ uptake into SR vesicles, mimicking the actions of the known SR Ca2+ channel blocker ruthenium red. The underlying mechanism of Fe(II) on SR Ca2+ transport is shown to be a direct and potent action on the ryanodine receptor. Fe(II) inhibits the binding of [3H]ryanodine when assayed in the presence of 5 microM Ca2+ with an IC50 of 4 microM and in an apparently cooperative manner (nH = 1.7). In the presence of physiological (1 mM) Mg2+, Fe(II) decreases the sensitivity of ryanodine receptors toward activation by Ca2+ shifting EC50 from 18 to 35 microM in the absence and presence of 5 microM Fe(II), respectively, without significant decrease in maximum [3H]ryanodine occupancy. In the presence of 5 microM Ca2+ and 1 mM Mg2+, Fe(II) decreases the potency of doxorubicin (DXR) on [3H]ryanodine binding (shifts EC50 from 8 to 24 microM in the absence and presence of 5 microM Fe(II)). These results suggest that Fe(II) competes with Ca2+ at the activator sites on the channel complex. The actions of Fe(II) on ryanodine receptor function is not correlated with membrane lipid peroxidation of SR vesicles since Fe(II) does not produce detectable changes in malondialdehyde using the thiobarbituric acid assay. These results demonstrate a direct inhibition of the Ca2+ release channel of cardiac SR by Fe(II) which may be important in pathological states of the heart during iron overload.

Topics: Animals; Binding, Competitive; Calcium; Calcium Channels; Chlorides; Doxorubicin; Ferric Compounds; Ferrous Compounds; Heart; Lipid Peroxidation; Male; Muscle Proteins; Myocardium; Rats; Rats, Sprague-Dawley; Ruthenium Red; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sarcoplasmic Reticulum