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Biomedical subjects

V E Kagan

Publications and source records attributed to V E Kagan.

At least 37 records · Page 2Linked to original sources
PubMed2001

Nitric oxide-dependent pro-oxidant and pro-apoptotic effect of metallothioneins in HL-60 cells challenged with cupric nitrilotriacetate.

Intracellular safeguarding functions of metallothioneins (MTs) include sequestering transition and heavy metals, scavenging free radicals and protecting against electrophiles. We report that MT protection against Cu-induced cytotoxicity can be reversed and pro-oxidant and pro-apoptotic effects can be induced in HL-60 cells exposed to NO. We demonstrate that in ZnCl(2)-pretreated HL-60 cells loaded with copper nitrilotriacetate (Cu-NTA), exposure to an NO donor, S-nitroso-N-acetyl penicillamine, resulted in S-nitrosylation and oxidation of MT cysteines. This disruption of MT Cu-binding thiolate clusters caused loosening and release of redox-active Cu, enhanced redox-cycling activity of Cu and increased peroxidation of major classes of membrane phospholipids. We also found that Cu-induced oxidative stress in ZnCl(2)-pretreated/Cu-NTA-loaded HL-60 cells was accompanied by apoptosis documented by characteristic changes of nuclear morphology, internucleosomal DNA cleavage, externalization of phosphatidylserine, release of cytochrome c from mitochondria into cytosol and activation of caspase-3. We conclude that in Cu-challenged cells, NO can reverse the protective role of MTs and convert them into pro-oxidant, pro-apoptotic implements.

Annexin A5↗
PubMed2001

Quantitative analysis of phospholipid peroxidation and antioxidant protection in live human epidermal keratinocytes.

To characterize oxidative stress in phospholipids of normal human epidermal keratinocytes we metabolically labeled their membrane phospholipids with a natural oxidation-sensitive fluorescent fatty acid, cis-parinaric acid, and exposed the cells to two different sources of oxidants--a lipid-soluble azo-initiator of peroxyl radicals, 2,2'-azobis(2,4-dimethyl-valeronitrile), AMVN, and a superoxide generator, xanthine oxidase/xanthine. We demonstrated that both oxidants induced pronounced oxidation of four major classes of cis-parinaric acid-labeled phospholipids-phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol-in normal human epidermal keratinocytes that was not detectable as any significant change of their phospholipid composition. Vitamin E was effective in protecting the cells against phospholipid peroxidation. Since viability of normal human epidermal keratinocytes was not changed either by labeling or exposure to oxidants the labeling protocol and oxidative stress employed are compatible with the quantitative analysis of phospholipid peroxidation in viable cells.

Adult↗
PubMed2001

Mishandling of copper by albumin: role in redox-cycling and oxidative stress in preeclampsia plasma.

OBJECTIVE: To test the hypothesis that enhanced oxidative stress during pregnancies complicated by preeclampsia is associated with improper copper (Cu) binding by plasma albumin, resulting in enhanced Cu redox-cycling activity and that altered Cu binding, in turn, is caused by interactions of excessive amounts of free fatty acids with albumin. STUDY DESIGN: We studied binding and redox-cycling activity of Cu in 17 normal pregnancy and 17 preeclampsia plasma samples. Binding of exogenous Cu in plasma samples was quantified indirectly using spectrophotometric measurements of its complex with a specific chelator of Cu(I), bathocuproine disulfonate. Redox-cycling activity of Cu in plasma samples was estimated by electron paramagnetic resonance (EPR) spectroscopy of ascorbate radicals formed during one-electron oxidation of ascorbate by redox-active catalytic Cu. Formation of Cu/albumin complexes in model systems in the presence and absence of fatty acids was studied using EPR spectroscopy of Cu(II)/albumin. RESULTS: We found that preeclampsia plasma (as compared to normal pregnancy plasma) (1) displays elevated endogenous ascorbate redox-cycling that is normalized by a Cu(II) chelator, cuprizone I, (2) has lowered capacity to bind and redox-regulate exogenously added Cu, and (3) responds to treatment with fatty-acid-free albumin by diminished ascorbate oxidizing activity. Conversely, addition of free fatty acid (oleic acid) to normal pregnancy plasma sample yields increased ascorbate redox-cycling activity. Our model experiments showed that Cu-dependent redox-cycling activity of purified human serum albumin is significantly increased by excess free fatty acids. CONCLUSION: Mishandling of Cu by albumin contributes to oxidative stress in preeclampsia. Cu chelators may represent promising mechanism-based antioxidants to attenuate oxidative stress in preeclampsia.

Adult↗
PubMed2001

Antioxidant mechanisms of nitric oxide against iron-catalyzed oxidative stress in cells.

Three distinct antioxidant pathways are considered through which iron-catalyzed oxidative stress may be regulated by nitric oxide (NO). The first two pathways involve direct redox interactions of NO with iron catalytic sites and represent a fast response that may be considered an emergency mechanism to protect cells from the consequences of acute and intensive oxidative stress. These are (i) NO-induced nitrosylation at heme and non-heme iron catalytic sites that is capable of directly reducing oxoferryl-associated radicals, (ii) formation of nitrosyl complexes with intracellular "loosely" bound redox-active iron, and (iii) an indirect regulatory pathway that may function as an adaptive mechanism that becomes operational upon long-term exposure of cells to NO. In the latter pathway, NO down-regulates expression of iron-containing proteins to prevent their catalytic prooxidant reactions.

Animals↗
PubMed2001

Modulation of redox signal transduction pathways in the treatment of cancer.

Reactive oxygen species (ROS)-mediated damage to DNA is associated with induction of stress-activated protein kinases leading to secondary and tertiary effects on the nuclear matrix, cytoplasmic transport mechanisms, and altered mitochondrial and cell membranes. The cellular defenses against ROS damage are associated with up-regulation of gene products that can significantly alter cell biology, including antiapoptotic Bax family proteins and inflammatory proteins. Altered cell integrity can occur either directly or by indirect paracrine and juxtacrine interactions within tissues. Previous approaches toward therapeutic intervention against ROS damage have included administration of radical scavenger compounds, use of novel drugs that increase cellular production of constitutive antioxidants, or pharmacologic agents that modify the intracellular transport of antioxidants. Strategies to modify the cellular effects of ROS in hyperbaric oxygen injury to the lung, reperfusion injury to transplanted organs, and cancer have led to novel approaches of gene therapy in which the transgenes for antioxidant proteins can be expressed in specific tissues. Reducing tissue-damaging effects of ROS may have relevance to cancer patients by ameliorating normal tissue damage from ionizing irradiation therapy, photodynamic therapy, and cancer chemotherapy.

Animals↗
PubMed2000

Quinolizin-coumarins as physical enhancers of chemiluminescence during lipid peroxidation in live HL-60 cells.

We investigated whether physical enhancers of low-level chemiluminescence-coumarin laser dyes C-314, C-334, and C-525--may be used to monitor interactions of lipid peroxyl radicals during lipid peroxidation in live cells. We present data demonstrating that two quinolizin-substituted coumarins--C-525 and C-334--can be integrated into HL-60 cells and successfully used as physical enhancers of chemiluminescence induced by the lipid soluble azo-initiator 2,2'-azobis(2,4-dimethyl-valeronitrile) (AMVN). Coumarins did not inhibit AMVN-induced peroxidation of membrane phospholipids in HL-60 cells, and no consumption of these coumarins occurred in the course of AMVN-induced oxidative stress. Redox status, evaluated by intracellular GSH content, remained unchanged after treatment with the coumarins. tert-Butyl hydroperoxide and cumene hydroperoxide (more hydrophilic oxidants) induced a lower chemiluminescence signal with both coumarins. Viability of HL-60 cells was not affected by coumarins both in the presence and in the absence of oxidants. Based on these results we conclude that quinolizin-substituted coumarins represent a promising class of physical enhancers of chemiluminescence for monitoring free radical peroxidation in live cells.

Azo Compounds↗
PubMed2000

Phospholipid signaling in apoptosis: peroxidation and externalization of phosphatidylserine.

The role of phospholipids in apoptosis signaling and the relationship between oxidation of phosphatidylserine and its redistribution in the plasma membrane were studied. A novel method for detection of site-specific phospholipid peroxidation based on the use of cis-parinaric acid as a reporter molecule metabolically integrated into membrane phospholipids in living cells was employed. When several tissue culture cell lines and different exogenous oxidants were used, the relationship between the oxidation of phosphatidylserine and apoptosis has been revealed. The plasma membrane was the preferred site of phosphatidylserine oxidation in cells. It was shown that selective oxidation of phosphatidylserine precedes its translocation from the inside to the outside surface of the plasma membrane during apoptosis. A model is proposed in which cytochrome c released from mitochondria by oxidative stress binds to phosphatidylserine located at the cytoplasmic surface of the plasma membrane and induces its oxidation. Interaction of peroxidized phosphatidylserine with aminophospholipid translocase causes inhibition of the enzyme relevant to phosphatidylserine externalization.

Animals↗
PubMed2000

Oxidative signaling pathway for externalization of plasma membrane phosphatidylserine during apoptosis.

Active maintenance of membrane phospholipid asymmetry is universal in normal cell membranes and its disruption with subsequent externalization of phosphatidylserine is a hallmark of apoptosis. Externalized phosphatidylserine appears to serve as an important signal for targeting recognition and elimination of apoptotic cells by macrophages, however, the molecular mechanisms responsible for phosphatidylserine translocation during apoptosis remain unresolved. Studies have focused on the function of aminophospholipid translocase and phospholipid scramblase as mediators of this process. Here we present evidence that unique oxidative events, represented by selective oxidation of phosphatidylserine, occur during apoptosis that could promote phosphatidylserine externalization. We speculate that selective phosphatidylserine oxidation could affect phosphatidylserine recognition by aminophospholipid translocase and/or directly result in enzyme inhibition. The potential interactions between the anionic phospholipid phosphatidylserine and the redox-active cationic protein effector of apoptosis, cytochrome c, are presented as a potential mechanism to account for selective oxidation of phosphatidylserine during apoptosis. Thus, cytochrome c-mediated phosphatidylserine oxidation may represent an important component of the apoptotic pathway.

Animals↗
PubMed2000

Myeloperoxidase-catalyzed phenoxyl radicals of vitamin E homologue, 2,2,5,7,8-pentamethyl- 6-hydroxychromane, do not induce oxidative stress in live HL-60 cells.

We used myeloperoxidase-containing HL-60 cells to generate phenoxyl radicals from nontoxic concentrations of a vitamin E homologue, 2,2, 5,7,8-pentamethyl-6-hydroxychromane (PMC) to test whether these radicals can induce oxidative stress in a physiological intracellular environment. In the presence of H(2)O(2), we were able to generate steady-state concentrations of PMC phenoxyl radicals readily detectable by EPR in viable HL-60 cells. In HL-60 cells pretreated with succinylacetone, an inhibitor of heme synthesis, a greater than 4-fold decrease in myeloperoxidase activity resulted in a dramatically decreased steady-state concentrations of PMC phenoxyl radicals hardly detectable in EPR spectra. We further conducted sensitive measurements of GSH oxidation and protein sulfhydryl oxidation as well as peroxidation in different classes of membrane phospholipids in HL-60 cells. We found that conditions compatible with the generation and detection of PMC phenoxyl radicals were not associated with either oxidation of GSH, protein SH-groups or phospholipid peroxidation. We conclude that PMC phenoxyl radicals do not induce oxidative stress under physiological conditions in contrast to their ability to cause lipid peroxidation in isolated lipoproteins in vitro.

Cell Survival↗
PubMed2000

Interaction between 6-hydroxydopamine and transferrin: "Let my iron go".

The dopamine analogue 6-hydroxydopamine (6-OHDA) is selectively toxic to catecholaminergic neurons. Because of its selectivity for neuroblastic cells in the sympathetic nervous system lineage, 6-OHDA has been suggested as a chemotherapeutic agent for targeted treatment of patients with neuroblastoma. We tested the hypothesis that the toxicity of 6-OHDA is caused by its interaction with serum ferric transferrin (Fe-TF) resulting in release of iron. We further hypothesized that this iron, through its redox-cycling by 6-OHDA, triggers generation of reactive oxygen species. 6-OHDA-induced release of iron from Fe-TF was demonstrated by: (1) low-temperature EPR spectroscopic evidence for decay of the characteristic Fe-TF signal (g = 4.3) and appearance of the high-spin signal from iron chelated by 6-OHDA oxidation products; (2) spectrophotometric detection of complexing of iron with the Fe(2+) chelator ferrozine; (3) redox-cycling of ascorbate yielding EPR-detectable ascorbate radicals; and (4) generation of hydroxyl radicals as evidenced by EPR spectroscopy of their adduct with a spin trap, 5, 5'-dimethylpyrroline oxide (DMPO) (DMPO-OH). Our low-temperature EPR studies showed that in human plasma, 6-OHDA caused iron release only under nitrogen gas but not under air or oxygen. The absence of a 6-OHDA effect in plasma under aerobic conditions was most likely due to its ferroxidase activity [with consequent reuptake of Fe(III) by apoTF] and catalytic oxidation of 6-OHDA by ceruloplasmin. Modeling of these plasma activities by a stable nitroxide radical, 2,2,6, 6-tetramethyl-1-piperidinyloxy (TEMPOL), resulted in protection of plasma Fe-TF against iron release under nitrogen. Parenteral administration of 6-OHDA to mice resulted in iron release from Fe-TF as evidenced by transformation of the Fe-TF low-temperature EPR signal that was indistinguishable from that seen in in vitro models. In addition, administration of the iron chelator deferoxamine (DFO) to mice prior to administration of toxic doses of 6-OHDA resulted in a decrease in activity impairment of mice as compared to that seen with 6-OHDA alone. These findings underscore the physiological and pharmacological relevance of 6-OHDA-mediated iron release from Fe-TF and suggest that iron chelators (DFO) may be used for prevention of 6-OHDA toxicity.

Animals↗
PubMed2000

Cell line dependence of Bcl-2-induced alteration of glutathione handling.

Bcl-2 has been associated with both oxidative and antioxidative effects in vivo. Moreover, despite evidence that Bcl-2 is antiapoptotic by virtue of its effect on reactive oxygen species and their scavengers, Bcl-2 exerts its antiapoptotic effects even under anaerobic conditions. The reasons for the variable relationship between Bcl-2 and reactive oxygen species are not clear. The present studies demonstrate that the impact of Bcl-2 on glutathione (GSH) metabolism is cell line-dependent. Bcl-2 overproduction in PC12 cells is associated with increased functional thiol reserves, increased reductive activation of chemotherapeutic prodrugs, and GSH accumulation after treatment with N-acetylcysteine. In contrast, Bcl-2-overproducing MCF-7 breast cancer cells demonstrate neither altered GSH handling nor potentiation of chemotherapeutic prodrug reduction. These findings indicate that the effects of Bcl-2 on GSH handling are millieu-dependent. This could account for the variable effects of Bcl-2 in in vivo systems. Furthermore, since our previous studies have demonstrated that reduction-dependent prodrugs may be useful chemotherapeutic agents against tumors that demonstrate altered GSH handling, screening in vitro for alteration of GSH handling may predict responsiveness of such tumors to these reduction-dependent agents.

Acetylcysteine↗
PubMed2000

Nitric oxide dissociates lipid oxidation from apoptosis and phosphatidylserine externalization during oxidative stress.

Oxidative stress in biological membranes can regulate various aspects of apoptosis, including phosphatidylserine (PS) externalization. It is not known, however, if the targets for these effects are lipids or proteins. Nitric oxide (NO), a bifunctional modulator of apoptosis, has both antioxidant and prooxidant potential. We report here that the NO donor PAPANONOate completely protected all phospholipids, including PS, from oxidation in HL-60 cells treated with 2,2'-azobis(2,4-dimethylisovaleronitrile) (AMVN), presumably via the ability of NO to react with lipid-derived peroxyl radicals and terminate the propagation of lipid peroxidation. PAPANONOate, however, had no effect on PS externalization or other markers of apoptosis following AMVN. Therefore, PS oxidation is not required for PS externalization during AMVN-induced apoptosis. PS externalization was accompanied by inhibition of aminophospholipid translocase (APT). NO potentiated AMVN inhibition of APT. Treatment with PAPANONOate alone produced modest (20%) inhibition of APT without PS externalization. NO did not reverse AMVN-induced oxidation of glutathione and protein thiols. We speculate that APT was sensitive to AMVN and/or NO via modification of protein thiols critical for functional activity. Therefore, the lipoprotective effects of NO were insufficient to prevent PS externalization and apoptosis following oxidative stress. Other targets such as protein thiols may be important redox-sensitive regulators of apoptosis initiation and execution. Thus, in the absence of significant peroxynitrite formation, NO's antioxidant effects are restricted to protection of lipids, while modification of protein substrates continues to occur.

Apoptosis↗
PubMed2000

Reconstitution of apo-superoxide dismutase by nitric oxide-induced copper transfer from metallothioneins.

Little is known about copper transfer from Cu-metallothionein (Cu-MT) to various target proteins, such as apo-SOD, and the potential role of redox mechanisms in this transfer. We studied Cu transfer from Cu-MT to apo/Zn-SOD in a cell-free model system and found that Cu(5)-MT and Cu(10)-MT were able to reconstitute SOD activity only in the presence of a nitric oxide donor, (Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium++ +-1,2-diolate (NOC-15). The percentage of reconstitution by Cu(5)-MT and Cu(10)-MT was 34 and 83%, respectively, compared with that reconstituted by free Cu alone. A Cu chelation assay using bathocuproine disulfonate (BCS) showed that NOC-15 induced release of free Cu from Cu(10)-MT but not from Cu(5)-MT. The transfer of Cu from Cu-MT to apo/Zn-SOD was not accompanied by enhanced Cu-dependent generation of ascorbate radicals or hydroxyl radicals as measured by EPR spectroscopy. We found a 70% decrease in the number of 2,2'-dithiodipyridine titratable SH groups on MT after incubation with NOC-15. Overall, our results suggest that Cu-MT could potentially function in a nitric oxide-dependent pathway for the delivery of Cu to apo-SOD in copper-challenged cells.

Animals↗
PubMed2000

Antioxidant and antiapoptotic function of metallothioneins in HL-60 cells challenged with copper nitrilotriacetate.

Antioxidant activity is believed to be an important intracellular function of metallothioneins (MT), yet the specific mechanisms of their antioxidant action are not known. Under conditions when cells are challenged with elevated concentrations of free copper as a result of metabolic disturbances or environmental and occupational exposures, MTs may be ideally suited for antioxidant function as effective copper chelators. In the study presented here, we tested this hypothesis using a recently established model of copper nitrilotriacetate-induced oxidative stress in HL-60 cells. Since copper-induced oxidative stress triggers apoptosis, we further investigated antiapoptotic function of MTs in HL-60 cells. Using a Sephadex G-75 chromatographic partial purification of MTs from cell homogenates with subsequent immuno-dot-blot assay, we showed that zinc pretreatment yielded a pronounced induction of MTs in HL-60 cells. We report that zinc-induced MTs were able to (i) completely bind intracellular copper, (ii) completely quench redox-cycling activity of copper, (iii) significantly inhibit copper-dependent oxidative stress in membrane phospholipids, and (iv) prevent copper-dependent apoptosis and its characteristic biochemical features (cytochrome c release from mitochondria into cytosol, caspase-3 activation, and externalization of phosphatidylserine in plasma membranes). In separate experiments, we used lung fibroblasts derived from MT1, MT2 knockout mice (MT(-)(/)(-)) and MT wild-type (MT(+/+)) mice. ZnCl(2) pretreatment resulted in a more than 10-fold induction of MTs in MT(+/+) cells, whereas the MT content in MT(-)(/)(-) cells remained low, at levels approximately 100-fold lower than in their MT wild-type counterparts. MT(-)(/)(-) cells were very sensitive to Cu-NTA and, most importantly, showed no response to ZnCl(2) pretreatment. In contrast, MT(+/+) cells were relatively more resistant to Cu-NTA, and this resistance was remarkably enhanced by ZnCl(2) pretreatment. Combined, our results demonstrate that metallothioneins function as effective antioxidants and an antiapoptotic mechanism in copper-challenged HL-60 cells.

Animals↗
PubMed2000

Oxidative stress following traumatic brain injury in rats: quantitation of biomarkers and detection of free radical intermediates.

Oxidative stress may contribute to many pathophysiologic changes that occur after traumatic brain injury. In the current study, contemporary methods of detecting oxidative stress were used in a rodent model of traumatic brain injury. The level of the stable product derived from peroxidation of arachidonyl residues in phospholipids, 8-epi-prostaglandin F(2alpha), was increased at 6 and 24 h after traumatic brain injury. Furthermore, relative amounts of fluorescent end products of lipid peroxidation in brain extracts were increased at 6 and 24 h after trauma compared with sham-operated controls. The total antioxidant reserves of brain homogenates and water-soluble antioxidant reserves as well as tissue concentrations of ascorbate, GSH, and protein sulfhydryls were reduced after traumatic brain injury. A selective inhibitor of cyclooxygenase-2, SC 58125, prevented depletion of ascorbate and thiols, the two major water-soluble antioxidants in traumatized brain. Electron paramagnetic resonance (EPR) spectroscopy of rat cortex homogenates failed to detect any radical adducts with a spin trap, 5,5-dimethyl-1-pyrroline N:-oxide, but did detect ascorbate radical signals. The ascorbate radical EPR signals increased in brain homogenates derived from traumatized brain samples compared with sham-operated controls. These results along with detailed model experiments in vitro indicate that ascorbate is a major antioxidant in brain and that the EPR assay of ascorbate radicals may be used to monitor production of free radicals in brain tissue after traumatic brain injury.

Animals↗
PubMed2000

Redox cycling of phenol induces oxidative stress in human epidermal keratinocytes.

A variety of phenolic compounds are utilized for industrial production of phenol-formaldehyde resins, paints, lacquers, cosmetics, and pharmaceuticals. Skin exposure to industrial phenolics is known to cause skin rash, dermal inflammation, contact dermatitis, leucoderma, and cancer promotion. The biochemical mechanisms of cytotoxicity of phenolic compounds are not well understood. We hypothesized that enzymatic one-electron oxidation of phenolic compounds resulting in the generation of phenoxyl radicals may be an important contributor to the cytotoxic effects. Phenoxyl radicals are readily reduced by thiols, ascorbate, and other intracellular reductants (e.g., NADH, NADPH) regenerating the parent phenolic compound. Hence, phenolic compounds may undergo enzymatically driven redox-cycling thus causing oxidative stress. To test the hypothesis, we analyzed endogenous thiols, lipid peroxidation, and total antioxidant reserves in normal human keratinocytes exposed to phenol. Using a newly developed cis-parinaric acid-based procedure to assay site-specific oxidative stress in membrane phospholipids, we found that phenol at subtoxic concentrations (50 microM) caused oxidation of phosphatidylcholine and phosphatidylethanolamine (but not of phosphatidylserine) in keratinocytes. Phenol did not induce peroxidation of phospholipids in liposomes prepared from keratinocyte lipids labeled by cis-parinaric acid. Measurements with ThioGlo-1 showed that phenol depleted glutathione but did not produce thiyl radicals as evidenced by our high-performance liquid chromatography measurements of GS.-5, 5-dimethyl1pyrroline N-oxide nitrone. Additionally, phenol caused a significant decrease of protein SH groups. Luminol-enhanced chemiluminescence assay demonstrated a significant decrease in total antioxidant reserves of keratinocytes exposed to phenol. Incubation of ascorbate-preloaded keratinocytes with phenol produced an electron paramagnetic resonance-detectable signal of ascorbate radicals, suggesting that redox-cycling of one-electron oxidation products of phenol, its phenoxyl radicals, is involved in the oxidative effects. As no cytotoxicity was observed in keratinocytes exposed to 50 microM or 500 microM phenol, we conclude that phenol at subtoxic concentrations causes significant oxidative stress.

Antioxidants↗
PubMed2000

Reversible thiol-dependent activation of ryanodine-sensitive Ca2+ release channel by etoposide (VP-16) phenoxyl radical.

Many phenolic compounds can act as antioxidants by donating a proton to peroxyl radicals and quenching lipid peroxidation. Phenoxyl radicals produced this way or from metabolism by peroxidases, tyrosinase, or mixed-function oxidases, however, may react with sulfhydryl groups of proteins and other endogenous thiols. In this regard, phenolic compounds may have cytotoxic potential instead of antioxidant effects. We employed the anticancer drug, etoposide (VP-16), as a model phenolic compound to study the sensitivity of ryanodine-sensitive Ca2+ channel (RyR) to VP-16 phenoxyl radicals. The combination of VP-16 and tyrosinase, used to generate the etoposide phenoxyl radical, produced marked Ca2+ release from Ca2+-loaded RyR-rich vesicles prepared from terminal cisternae fraction of sarcoplasmic reticulum (SR). This effect was reversed by the SH-reagent, dithiothreitol (DTT), suggesting that cysteines within the RyR-protein complex were targets for modification by VP-16 phenoxyl radicals. VP-16/tyrosinase-induced release of Ca2+ was attenuated in vesicles prepared from longitudinal SR, which contain relatively little RyR. The effects of the VP-16 phenoxyl radical on Ca2+-ATPase in SR vesicles resembled those observed with caffeine or 4,4'-dithiodipyridine, both of which activate RyR Ca2+ release and lead to activation of Ca2+-ATPase via prolonged Ca2+ cycling. The addition of ruthenium red returned Ca2+-ATPase to its original level. Thus, under these conditions Ca2+-ATPase was not directly affected by VP-16 phenoxyl radical. The hypersensitive SH-groups on RyR are shown to be targets for oxidation of VP-16 phenoxyl radical, and suggest that other phenolic compounds could similarly disrupt Ca2+ homeostasis.

Adenosine Triphosphate↗