Rabbit sarcoplasmic reticulum Ca(2+)-ATPase replaces yeast PMC1 and PMR1 Ca(2+)-ATPases for cell viability and calcineurin-dependent regulation of calcium tolerance

SERCA1a, the fast-twitch skeletal muscle isoform of sarco(endo)plasmic reticulum Ca(2+)-ATPase, was expressed in yeast using the promoter of the plasma membrane H(+)-ATPase. In the yeast Saccharomyces cerevisiae, the Golgi PMR1 Ca(2+)-ATPase and the vacuole PMC1 Ca(2+)-ATPase function together in Ca2+ sequestration and Ca2+ tolerance. SERCA1a expression restored growth of pmc1 mutants in media containing high Ca2+ concentrations, consistent with increased Ca2+ uptake in an internal compartment. SERCA1a expression also prevented synthetic lethality of pmr1 pmc1 double mutants on standard media. Electron microscopy and subcellular fractionation analysis showed that SERCA1a was localized in intracellular membranes derived from the endoplasmic reticulum. Finally, we found that SERCA1a ATPase activity expressed in yeast was regulated by calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase. This result indicates that calcineurin contributes to calcium homeostasis by modulating the ATPase activity of Ca2+ pumps localized in intra-cellular compartments.     

Peroxynitrite resistance of sarco/endoplasmic reticulum Ca2+ pump in pig coronary artery endothelium and smooth muscle

We examined the effects of peroxynitrite pre-treatment on sarco/endoplasmic reticulum Ca(2+) (SERCA) pump in pig coronary artery smooth muscle and endothelium. In saponin-permeabilized cells, smooth muscle showed much greater rates of the SERCA Ca(2+) pump-dependent (45)Ca(2+) uptake/mg protein than did the endothelial cells. Peroxynitrite treatment of cells inhibited the SERCA pump more severely in smooth muscle cells than in endothelial cells. To determine implications of this observation, we next examined the effect of the SERCA pump inhibitor cyclopiazonic acid (CPA) on intracellular Ca(2+) concentration of intact cultured cells. CPA produced cytosolic Ca(2+) transients in cultured endothelial and smooth muscle cells. Pre-treatment with peroxynitrite (200 microM) inhibited the Ca(2+) transients in the smooth muscle but not in the endothelial cells. CPA contracts de-endothelialized artery rings and relaxes precontracted arteries with intact endothelium. Peroxynitrite (250 microM) pre-treatment inhibited contraction in the de-endothelialized artery rings, but not the endothelium-dependent relaxation. Thus, endothelial cells appear to be more resistant than smooth muscle to the effects of peroxynitrite at the levels of SERCA pump activity, CPA-induced Ca(2+) transients in cultured cells, and the effects of CPA on contractility. The greater resistance of endothelium to peroxynitrite may play a protective role in pathological conditions such as ischemia-reperfusion when excess free radicals are produced.     

Complex effects of ryanodine on the sarcoplasmic reticulum Ca2+ levels in smooth muscle cells

We have studied the effects of ryanodine and inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) with thapsigargin, on both [Ca(2+)](i) and the sarcoplasmic reticulum (SR) Ca(2+) level during caffeine-induced Ca(2+) release in single smooth muscle cells. Incubation with 10 microM ryanodine did not inhibit the first caffeine-induced [Ca(2+)](i) response, although it abolished the [Ca(2+)](i) response to a second application of caffeine. To assess whether ryanodine was inducing a permanent depletion of the internal Ca(2+) stores, we measured the SR Ca(2+) level with Mag-Fura-2. The magnitude of the caffeine-induced reduction in the SR Ca(2+) level was not augmented by incubating cells with 1 microM ryanodine. Moreover, on removal of caffeine, the SR Ca(2+) levels partially recovered in 61% of the cells due to the activity of thapsigargin-sensitive SERCA pumps. Unexpectedly, 10 microM ryanodine instead of inducing complete depletion of SR Ca(2+) stores markedly reduced the caffeine-induced SR Ca(2+) response. It was necessary to previously inhibit SERCA pumps with thapsigargin for ryanodine to be able to induce caffeine-triggered permanent depletion of SR Ca(2+) stores. These data suggest that the effect of ryanodine on smooth muscle SR Ca(2+) stores was markedly affected by the activity of SERCA pumps. Our data highlight the importance of directly measuring SR Ca(2+) levels to determine the effect of ryanodine on the internal Ca(2+) stores.     

Vectorial Ca2+ release via ryanodine receptors contributes to Ca2+ extrusion from freshly isolated rabbit aortic endothelial cells

In this study, we identified ryanodine receptors (RyRs) as a component of a cytosolic Ca(2+) removal pathway in freshly isolated rabbit aortic endothelial cells. In an earlier article, we reported that the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and Na(+)/Ca(2+) exchanger (NCX) function in series to extrude cytosolic Ca(2+) to the extracellular space. Here we employed caffeine and ryanodine as modulators of RyR and showed that they act as the linkage between SERCA and NCX in removing Ca(2+) from the cytoplasm. Our data indicate that both 15 mM caffeine and 1 microM ryanodine facilitated Ca(2+) extrusion by activating RyRs while 100 microM ryanodine had the opposite effect by blocking RyRs. A further attempt to investigate RyR pharmacology revealed that in the absence of extracellular Ca(2+), ryanodine at 1 microM, but not 100 microM, stimulated Ca(2+) loss from the endoplasmic reticulum (ER). Blockade of RyR had no effect on the Ca(2+) removal rate when NCX had been previously blocked. In addition, the localization of RyR was determined using confocal microscopy of BODIPY TR-X fluorescent staining. Taken together, our findings suggest that in freshly isolated endothelial cells Ca(2+) is removed in part by transport through SERCA, RyR, and eventually NCX, and that RyR and NCX are in close functional proximity near the plasma membrane. After blockade of this component, Ca(2+) extrusion could be further inhibited by carboxyeosin, indicating a parallel contribution by the plasmalemmal Ca(2+)-ATPase (PMCA).     

SERCA pump optimizes Ca2+ release by a mechanism independent of store filling in smooth muscle cells

Thapsigargin-sensitive sarco/endoplasmic reticulum Ca(2+) pumps (SERCAs) are involved in maintaining and replenishing agonist-sensitive internal stores. Although it has been assumed that release channels act independently of SERCA pumps, there are data suggesting the opposite. Our aim was to study the relationship between SERCA pumps and the release channels in smooth muscle cells. To this end, we have rapidly blocked SERCA pumps with thapsigargin, to avoid depletion of the internal Ca(2+) stores, and induced Ca(2+) release with either caffeine, to open ryanodine receptors, or acetylcholine, to open inositol 1,4,5-trisphosphate receptors. Blocking SERCA pumps produced smaller and slower agonist-induced [Ca(2+)](i) responses. We determined the Ca(2+) level of the internal stores both indirectly, measuring the frequency of spontaneous transient outward currents, and directly, using Mag-Fura-2, and demonstrated that the inhibition of SERCA pumps did not produce a reduction of the sarco/endoplasmic reticulum Ca(2+) levels to explain the decrease in the agonist-induced Ca(2+) responses. It appears that SERCA pumps are involved in sustaining agonist-induced Ca(2+) release by a mechanism that involves the modulation of Ca(2+) availability in the lumen of the internal stores.     

Nucleoplasmic Ca(2+)loading is regulated by mobilization of perinuclear Ca(2+)

Regulation of nucleoplasmic calcium (Ca(2+)) concentration may occur by the mobilization of perinuclear luminal Ca(2+)pools involving specific Ca(2+)pumps and channels of both inner and outer perinuclear membranes. To determine the role of perinuclear luminal Ca(2+), we examined freshly cultured 10 day-old embryonic chick ventricular cardiomyocytes. We obtained evidence suggesting the existence of the molecular machinery required for the bi-directional Ca(2+)fluxes using confocal imaging techniques. Embryonic cardiomyocytes were probed with antibodies specific for ryanodine-sensitive Ca(2+)channels (RyR2), sarco/endoplasmic reticulum Ca(2+)ATPase (SERCA2)-pumps, and fluorescent BODIPY derivatives of ryanodine and thapsigargin. Using immunocytochemistry techniques, confocal imaging showed the presence of RyR2 Ca(2+)channels and SERCA2-pumps highly localized to regions surrounding the nucleus, referable to the nuclear envelope. Results obtained from Fluo-3, AM loaded ionomycin-perforated embryonic cardiomyocytes demonstrated that gradual increases of extranuclear Ca(2+)from 100 to 1600 nM Ca(2+)was localized to the nucleus. SERCA2-pump inhibitors thapsigargin and cyclopiazonic acid showed a concentration-dependent inhibition of nuclear Ca(2+)loading. Furthermore, ryanodine demonstrated a biphasic concentration-dependence upon active nuclear Ca(2+)loading. The concomitant addition of thapsigargin or cyclopiazonic acid with ryanodine at inhibitory concentrations caused an significant increase in nuclear Ca(2+)loading at low concentrations of extranuclear added Ca(2+). Our results show that the perinuclear lumen in embryonic chick ventricular cardiomyocytes is capable of autonomously regulating nucleoplasmic Ca(2+)fluxes.     

Modeling Ca2+ dynamics of mouse cardiac cells points to a critical role of SERCA's affinity for Ca2+

The SERCA2a isoform of the sarco/endoplasmic reticulum Ca(2+) pumps is specifically expressed in the heart, whereas SERCA2b is the ubiquitously expressed variant. It has been shown previously that replacement of SERCA2a by SERCA2b in mice (SERCA2(b/b) mice) results in only a moderate functional impairment, whereas SERCA activity is decreased by a 40% lower SERCA protein expression and by increased inhibition by phospholamban. To find out whether the documented kinetic differences in SERCA2b relative to SERCA2a (i.e., a twofold higher apparent Ca(2+) affinity, but twofold lower maximal turnover rate) can explain these compensatory changes, we simulated Ca(2+) dynamics in mouse ventricular myocytes. The model shows that the relative Ca(2+) transport capacity of SERCA2a and SERCA2b depends on the SERCA concentration. The simulations point to a dominant effect of SERCA2b's higher Ca(2+) affinity over its lower maximal turnover rate. The results suggest that increased systolic and decreased diastolic Ca(2+) levels in unstimulated conditions could contribute to the downregulation of SERCA in SERCA2(b/b) mice. In stress conditions, Ca(2+) handling is less efficient by SERCA2b than by SERCA2a, which might contribute to the observed hypertrophy in SERCA2(b/b) mice. Altogether, SERCA2a might be a better compromise between performance in basal conditions and performance during β-adrenergic stress.     

Ca(2+) removal mechanisms in freshly isolated rabbit aortic endothelial cells

Calcium removal from the cytoplasm was investigated in freshly isolated aortic endothelial cells by monitoring changes in intracellular calcium ([Ca(2+)](i)) using ratiometric fura-2 fluorimetry. Blockade of the Na(+)/Ca(2+) exchanger (NCX) by replacement of external sodium with equi-molar N-methyl-D-glutamine (0Na PSS) decreased the removal rate by 52%. Blockade of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) by cyclopiazonic acid (CPA) decreased the removal rate by 50%. Simultaneous application of CPA and 0Na PSS did not reduce the removal rate any further (53%). The lack of additivity of these two procedures, suggests that SERCA and the NCX function in series to lower [Ca(2+)](i). In addition, in the absence of extracellular Ca(2+), removal of external Na(+) markedly reduced the rate of loss of Ca(2+) from the ER further supporting the hypothesis that NCX is functionally linked to ER calcium release channels, and thus, plays an important role in ER calcium unloading. To investigate the mechanism for the coupling of NCX and SERCA, the same protocols as described above were repeated after treating the cells with cytochalasin D, which disrupts the cytoskeleton. This treatment uncoupled the NCX from SERCA, as evidenced by the resulting additive inhibitory effects of application of CPA and removal of extracellular Na(+) on the rate of Ca(2+) removal from the cytoplasm. These data suggest that in endothelial cells NCX and SERCA function in series to remove about half of the free Ca(2+) from the cytosol, while PMCA contributes to the other half of the Ca(2+) removal process.     

Cytosolic phosphorylation of calnexin controls intracellular Ca(2+) oscillations via an interaction with SERCA2b

Calreticulin (CRT) and calnexin (CLNX) are lectin chaperones that participate in protein folding in the endoplasmic reticulum (ER). CRT is a soluble ER lumenal protein, whereas CLNX is a transmembrane protein with a cytosolic domain that contains two consensus motifs for protein kinase (PK) C/proline- directed kinase (PDK) phosphorylation. Using confocal Ca(2+) imaging in Xenopus oocytes, we report here that coexpression of CLNX with sarco endoplasmic reticulum calcium ATPase (SERCA) 2b results in inhibition of intracellular Ca(2+) oscillations, suggesting a functional inhibition of the pump. By site-directed mutagenesis, we demonstrate that this interaction is regulated by a COOH-terminal serine residue (S562) in CLNX. Furthermore, inositol 1,4,5-trisphosphate- mediated Ca(2+) release results in a dephosphorylation of this residue. We also demonstrate by coimmunoprecipitation that CLNX physically interacts with the COOH terminus of SERCA2b and that after dephosphorylation treatment, this interaction is significantly reduced. Together, our results suggest that CRT is uniquely regulated by ER lumenal conditions, whereas CLNX is, in addition, regulated by the phosphorylation status of its cytosolic domain. The S562 residue in CLNX acts as a molecular switch that regulates the interaction of the chaperone with SERCA2b, thereby affecting Ca(2+) signaling and controlling Ca(2+)-sensitive chaperone functions in the ER.     

Ca2+-transport in sea urchin unfertilized eggs: regulation by endogenous sulfated polysaccharides and K+

Previous data from our laboratory showed that the reticulum of the sea cucumber smooth muscle body wall retains both a sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) and a sulfated polysaccharide. In this invertebrate, the transport of Ca(2+) by the SERCA is naturally inhibited by these endogenous sulfated polysaccharides. The inhibition is reverted by K(+) leading to an enhancement of the Ca(2+) transport rate. We now show that vesicles derived from the endoplasmic reticulum of unfertilized eggs from the sea urchin Arbacia lixula retain a SERCA that is able to transport Ca(2+) at the expense of ATP hydrolysis. As described for the sea cucumber SERCA isoform, the enzyme from the sea urchin is activated by K(+) but not by Li(+) and is inhibited by thapsigargin, a specific inhibitor of SERCA. A new sulfated polysaccharide was identified in the sea urchin eggs reticulum composed mainly by galactose, glucose, hexosamine and manose. After extraction and purification, this sulfated polysaccharide was able to inhibit the mammal SERCA isoform found in rabbit skeletal muscle and the inhibition is reversed by K(+). These data suggest that the regulation of the SERCA pump by K(+) and sulfated polysaccharides is not restricted to few marine invertebrates but is widespread.     

Sarco/endoplasmic reticulum Ca2+ (SERCA)-pumps: link to heart beats and calcium waves.

Mobilization of endoplasmic reticulum Ca2+ is pivotal to the ability of a cell to send or respond to stimuli. Ca(2+)-Mg(2+)-ATPases, termed SERCA pumps, sequester Ca2+ into the sarco/endoplasmic reticulum. There are several SERCA protein isoforms encoded by three genes. This paper summarizes the structure, function, tissue and subcellular distribution, and regulation of various SERCA isoforms. Then it attempts to link divergence in the signal transduction processes of cells to the types and levels of SERCA proteins they express and to how the cells regulate their SERCA pump activity. The paper examines possible linkages between SERCA pumps and receptor-activated Ca2+ entry, SERCA isoform localization and Ca(2+)-waves, and the role of SERCA pumps in nuclear Ca2+ in cell proliferation and apoptosis. Then it uses available information on cardiac function and chronic stimulation of the fast-twitch muscle to answer a series of basic questions on the regulation of SERCA activity and expression and their linkage to signal transduction. Finally, it discusses the possibility that neurons exhibit complex Ca(2+)-waves whose interactions have the potential to explain the operational basis of neural networks. A series of unanswered questions emerge based on this synthesis, including the unsettling issue of whether all the isoforms are needed to achieve the divergence in signal transduction or if there is a degree of redundancy in the system.     

S-Glutathiolation by peroxynitrite activates SERCA during arterial relaxation by nitric oxide

Nitric oxide (NO) physiologically stimulates the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) to decrease intracellular Ca(2+) concentration and relax cardiac, skeletal and vascular smooth muscle. Here, we show that NO-derived peroxynitrite (ONOO(-)) directly increases SERCA activity by S-glutathiolation and that this modification of SERCA is blocked by irreversible oxidation of the relevant cysteine thiols during atherosclerosis. Purified SERCA was S-glutathiolated by ONOO(-) and the increase in Ca(2+)-uptake activity of SERCA reconstituted in phospholipid vesicles required the presence of glutathione. Mutation of the SERCA-reactive Cys674 to serine abolished these effects. Because superoxide scavengers decreased S-glutathiolation of SERCA and arterial relaxation by NO, ONOO(-) is implicated as the intracellular mediator. NO-dependent relaxation as well as S-glutathiolation and activation of SERCA were decreased by atherosclerosis and Cys674 was found to be oxidized to sulfonic acid. Thus, irreversible oxidation of key thiol(s) in disease impairs NO-induced relaxation by preventing reversible S-glutathiolation and activation of SERCA by NO/ONOO(-).     

Temperature dependence of the Ca2+-ATPase (SERCA2) in the ventricles of tuna and mackerel

Recent physiological studies on the cardiovascular performance of tunas suggest that the elevated heart rates of these fish may rely on increased use of intracellular sarcoplasmic reticulum (SR) Ca2+ stores. In this study, we compare the cellular cardiac performance in endothermic tunas (bluefin, albacore, yellowfin) and their ectothermic sister taxa (mackerel) in response to acute temperature change. The cardiac sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2) plays a major role during cardiac excitation-contraction (E-C) coupling, transporting Ca2+ from the cytosol into the lumen of the SR and thus promoting the relaxation of the muscle. Measurements of oxalate-supported Ca2+ uptake in SR-enriched ventricular vesicles indicated that tunas were capable of sustaining a rate of Ca2+ uptake that was significantly higher than the mackerel. Among tunas, the cold-tolerant bluefin had the highest rates of SR Ca2+ uptake and ATPase activity. The differences among Ca2+ uptake and ATP hydrolysis rates do not seem to result from intrinsic differences between the SERCA2 present in the different tunas, as shown by their similar temperature sensitivities and similar values for activation energy. Western blots reveal that increased SERCA2 protein content is associated with the higher Ca2+ uptake and ATPase activities seen in bluefin ventricles compared with albacore, yellowfin, and mackerel. We hypothesize that a key step in the evolution of high heart rate and high metabolic rate in tunas is increased activity of the SERCA2 enzyme. We also suggest that high levels of SERCA2 in bluefin tuna hearts may be important for retaining cardiac function at cold temperatures.     

Intracellular alkalinization induces cytosolic Ca2+ increases by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)

Intracellular pH (pHi) and Ca(2+) regulate essentially all aspects of cellular activities. Their inter-relationship has not been mechanistically explored. In this study, we used bases and acetic acid to manipulate the pHi. We found that transient pHi rise induced by both organic and inorganic bases, but not acidification induced by acid, produced elevation of cytosolic Ca(2+). The sources of the Ca(2+) increase are from the endoplasmic reticulum (ER) Ca(2+) pools as well as from Ca(2+) influx. The store-mobilization component of the Ca(2+) increase induced by the pHi rise was not sensitive to antagonists for either IP(3)-receptors or ryanodine receptors, but was due to inhibition of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), leading to depletion of the ER Ca(2+) store. We further showed that the physiological consequence of depletion of the ER Ca(2+) store by pHi rise is the activation of store-operated channels (SOCs) of Orai1 and Stim1, leading to increased Ca(2+) influx. Taken together, our results indicate that intracellular alkalinization inhibits SERCA activity, similar to thapsigargin, thereby resulting in Ca(2+) leak from ER pools followed by Ca(2+) influx via SOCs.     

Expression of SERCA isoform with faster Ca2+ transport properties improves postischemic cardiac function and Ca2+ handling and decreases myocardial infarction

Myocardial ischemia-reperfusion (I/R) injury is associated with contractile dysfunction, arrhythmias, and myocyte death. Intracellular Ca(2+) overload with reduced activity of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) is a critical mechanism of this injury. Although upregulation of SERCA function is well documented to improve postischemic cardiac function, there are conflicting reports where pharmacological inhibition of SERCA improved postischemic function. SERCA2a is the primary cardiac isoform regulating intracellular Ca(2+) homeostasis; however, SERCA1a has been shown to substitute SERCA2a with faster Ca(2+) transport kinetics. Therefore, to further address this issue and to evaluate whether SERCA1a expression could improve postischemic cardiac function and myocardial salvage, in vitro and in vivo myocardial I/R studies were performed on SERCA1a transgenic (SERCA1a(+/+)) and nontransgenic (NTG) mice. Langendorff-perfused hearts were subjected to 30 min of global ischemia followed by reperfusion. Baseline preischemic coronary flow and left ventricular developed pressure were significantly greater in SERCA1a(+/+) mice compared with NTG mice. Independent of reperfusion-induced oxidative stress, SERCA1a(+/+) hearts demonstrated greatly improved postischemic (45 min) contractile recovery with less persistent arrhythmias compared with NTG hearts. Morphometry showed better-preserved myocardial structure with less infarction, and electron microscopy demonstrated better-preserved myofibrillar and mitochondrial ultrastructure in SERCA1a(+/+) hearts. Importantly, intraischemic Ca(2+) levels were significantly lower in SERCA1a(+/+) hearts. The cardioprotective effect of SERCA1a was also observed during in vivo regional I/R with reduced myocardial infarct size after 24 h of reperfusion. Thus SERCA1a(+/+) hearts were markedly protected against I/R injury, suggesting that expression of SERCA 1a isoform reduces postischemic Ca(2+) overload and thus provides potent myocardial protection.     

Ca2+-dependent redox modulation of SERCA 2b by ERp57

We demonstrated previously that calreticulin (CRT) interacts with the lumenal COOH-terminal sequence of sarco endoplasmic reticulum (ER) calcium ATPase (SERCA) 2b to inhibit Ca2+ oscillations. Work from other laboratories demonstrated that CRT also interacts with the ER oxidoreductase, ER protein 57 (also known as ER-60, GRP58; ERp57) during folding of nascent glycoproteins. In this paper, we demonstrate that ERp57 overexpression reduces the frequency of Ca2+ oscillations enhanced by SERCA 2b. In contrast, overexpression of SERCA 2b mutants defective in cysteines located in intralumenal loop 4 (L4) increase Ca2+ oscillation frequency. In vitro, we demonstrate a Ca2+-dependent and -specific interaction between ERp57 and L4. Interestingly, ERp57 does not affect the activity of SERCA 2a or SERCA 2b mutants lacking the CRT binding site. Overexpression of CRT domains that disrupt the interaction of CRT with ERp57 behave as dominant negatives in the Ca2+ oscillation assay. Our results suggest that ERp57 modulates the redox state of ER facing thiols in SERCA 2b in a Ca2+-dependent manner, providing dynamic control of ER Ca2+ homeostasis.     

Exposure of aconitase to smoking-related oxidants results in iron loss and increased iron response protein-1 activity: potential mechanisms for iron accumulation in human arterial cells

Smokers have an elevated risk of cardiovascular disease, but the origin(s) of this increased risk are incompletely defined. Evidence supports an accumulation of the oxidant-generating enzyme myeloperoxidase (MPO) in the inflamed artery wall, and smokers have high levels of SCN^?, a preferred MPO substrate, with this resulting in HOSCN formation. We hypothesised that HOSCN, a thiol-specific oxidant may target the iron-sulphur cluster of aconitase (both isolated, and within primary human coronary artery endothelial cells; HCAEC) resulting in enzyme dysfunction, release of iron, and conversion of the cytosolic isoform to iron response protein-1, which regulates intracellular iron levels. We show that exposure of isolated aconitase to increasing concentrations of HOSCN releases iron from the aconitase [Fe-S]₄ cluster, and decreases enzyme activity. This is associated with protein thiol loss and modification of specific Cys residues in, and around, the [Fe-S]₄ cluster. Exposure of HCAEC to HOSCN resulted in increased intracellular levels of chelatable iron, loss of aconitase activity and increased iron response protein-1 (IRP-1) activity. These data indicate HOSCN, an oxidant associated with oxidative stress in smokers, can induce aconitase dysfunction in human endothelial cells via Cys oxidation, damage to the [Fe-S]₄ cluster, iron release and generation of IRP-1 activity, which modulates ferritin protein levels and results in dysregulation of iron metabolism. These data may rationalise, in part, the presence of increased levels of iron in human atherosclerotic lesions and contribute to increased oxidative damage and endothelial cell dysfunction in smokers. Similar reactions may occur at other sites of inflammation.     

Baseline cytosolic Ca2+ oscillations derived from a non-endoplasmic reticulum Ca2+ store

Cytosolic Ca(2+) oscillations can be due to cycles of release and re-uptake of internally stored Ca(2+). To investigate the nature of these Ca(2+) stores, we expressed the Pmr1 Ca(2+) pump of Caenorhabditis elegans in COS-1 cells and pretreated the cells with thapsigargin to prevent Ca(2+) uptake by the sarco(endo)plasmic reticulum Ca(2+)-ATPase. Pmr1 co-localized with the Golgi-specific 58K protein and was targeted to a Ca(2+) store that was less leaky for Ca(2+) than the endoplasmic reticulum and whose inositol trisphosphate receptors were less sensitive to inositol trisphosphate and ATP than those in the endoplasmic reticulum. ATP-stimulated Pmr1-overexpressing cells responded after a latency to extracellular Ca(2+) with a regenerative Ca(2+) signal, which could be prevented by caffeine. They also produced very stable ilimaquinone-sensitive baseline Ca(2+) spikes, even in the presence of thapsigargin. Such responses never occurred in non-transfected cells or in cells that overexpressed the type-1 sarco(endo)plasmic reticulum Ca(2+)-ATPase. Abortive Ca(2+) spikes also occurred in histamine-stimulated untransfected HeLa cells pretreated with thapsigargin, and they too were inhibited by ilimaquinone. We conclude that the Pmr1-induced Ca(2+) store, which probably corresponds to the Golgi compartment, can play a crucial role in setting up baseline Ca(2+) spiking.     

3-Bromopyruvate inhibits calcium uptake by sarcoplasmic reticulum vesicles but not SERCA ATP hydrolysis activity

3-Bromopyruvate (3BrPA) is an antitumor agent that alkylates the thiol groups of enzymes and has been proposed as a treatment for neoplasias because of its specific reactivity with metabolic energy transducing enzymes in tumor cells. In this study, we show that the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) type 1 is one of the target enzymes of 3BrPA activity. Sarco/endoplasmic reticulum vesicles (SRV) were incubated in the presence of 1mM 3BrPA, which was unable to inhibit the ATPase activity of SERCA. However, Ca(2+)-uptake activity was significantly inhibited by 80% with 150 μM 3BrPA. These results indicate that 3BrPA has the ability to uncouple the ATP hydrolysis from the calcium transport activities. In addition, we observed that the inclusion of 2mM reduced glutathione (GSH) in the reaction medium with different 3BrPA concentrations promoted an increase in 40% in ATPase activity and protects the inhibition promoted by 3BrPA in calcium uptake activity. This derivatization is accompanied by a decrease of reduced cysteine (Cys), suggesting that GSH and 3BrPA increases SERCA activity and transport by pyruvylation and/or S-glutathiolation mediated by GSH at a critical Cys residues of the SERCA.     

The Ca2+/Mn2+ pumps in the Golgi apparatus

Recent evidence highlights the functional importance of the Golgi apparatus as an agonist-sensitive intracellular Ca(2+) store. Besides Ca(2+)-release channels and Ca(2+)-binding proteins, the Golgi complex contains Ca(2+)-uptake mechanisms consisting of the well-known sarco/endoplasmic reticulum Ca(2+)-transport ATPases (SERCA) and the much less characterized secretory-pathway Ca(2+)-transport ATPases (SPCA). SPCA supplies the Golgi compartments and, possibly, the more distal compartments of the secretory pathway with both Ca(2+) and Mn(2+) and, therefore, plays an important role in the cytosolic and intra-Golgi Ca(2+) and Mn(2+) homeostasis. Mutations in the human gene encoding the SPCA1 pump (ATP2C1) resulting in Hailey-Hailey disease, an autosomal dominant skin disorder, are discussed.