Nmol Ca2 Research Articles

Calpain I Induces Cleavage and Release of Apoptosis-inducing Factor from Isolated Mitochondria

The translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus has been implicated in the mechanism of glutamate excitotoxicity in cortical neurons and has been observed in vivo following acute rodent brain injuries. However, the mechanism and time course of AIF redistribution to the nucleus is highly controversial. Because elevated intracellular calcium is one of the most ubiquitous features of neuronal cell death, this study tested the hypothesis that cleavage of AIF by the calcium-activated protease calpain mediates its release from mitochondria. Both precursor and mature forms of recombinant AIF were cleaved near the amino terminus by calpain I in vitro. Mitochondrial outer membrane permeabilization by truncated Bid induced cytochrome c release from isolated liver or brain mitochondria but only induced AIF release in the presence of active calpain. Enzymatic inhibition of calpain by calpeptin precluded AIF release, demonstrating that proteolytic activity was required for release. Calpeptin and the mitochondrial permeability transition pore antagonist cyclosporin A also inhibited calcium-induced AIF release from mouse liver mitochondria, implicating the involvement of an endogenous mitochondrial calpain in release of AIF during permeability transition. Cleavage of AIF directly decreased its association with pure lipid vesicles of mitochondrial inner membrane composition. Taken together, these results define a novel mechanism of AIF release involving calpain processing and identify a potential molecular checkpoint for cytoprotective interventions.

Reduced sarcoplasmic reticulum Ca2+ uptake and increased Na+?Ca2+ exchanger expression in left ventricle myocardium of dogs with progression of heart failure

Alteration of intracellular Ca(2+) homeostasis in failing cardiomyocytes is associated with changes in regulatory proteins located in the sarcoplasmic reticulum (SR) and sarcolemma, which participate in Ca(2+) fluxes across the membrane during the cardiac cycle. These regulatory proteins include Ca(2+)-ATPase (SERCA 2A), phospholamban (PLB), ryanodine-sensitive Ca(2+) release channels (RR), and the sarcolemmal Na(+)-Ca(2+) exchanger (NCX). Although their status is known in failed myocardium, it is poorly understood during the progression of heart failure (HF), particularly in large animals. We studied the left ventricular (LV) myocardium of six dogs with moderate HF and six with severe HF produced by multiple intracoronary microembolizations, compared with six normal dogs (NL). Oxalate-dependent SR Ca(2+) uptake and expression of SERCA 2A, PLB, phosphorylated PLB at serine 16 (PLB-Ser) and threonine 17 (PLB-Thr), RR, and NCX were determined. Percent LV ejection fraction declined by 47% compared with NL (34.1% +/- 1% vs 64% +/- 2%) in dogs with moderate HF (HF-2W) 2 weeks after the last embolization and by 42% (20.5% +/- 1% vs 34.1% +/- 1%) in dogs with severe HF (HF-4M) at 4 months compared with HF-2W. Left ventricular pressure during isovolumic contraction (+dP/dt, mmHg/s) and relaxation (-dP/dt, mmHg/s) was significantly reduced in severe compared with moderate HF. Oxalate-dependent SR Ca(2+) uptake (nmol (45)Ca(2+) accumulated/min per milligram noncollagen protein) declined by 25% (21.3 +/- 1 vs 28.5 +/- 2) in HF-2W and 49% in HF-4M. Protein expression of SERCA 2A and PLB decreased by 67% and 35%, respectively, in HF-2W compared with NL, whereas SERCA 2A expression increased by 167% and PLB decreased by 40% in HF-4M compared with HF-2W. However, SERCA 2A protein was still significantly lower in HF-4M compared with NL. PLB-Ser and PLB-Thr increased significantly in HF-2W but decreased in HF-4M compared with NL. Similar changes in mRNA encoding PLB and SERCA 2A were observed in dogs with moderate and severe HF. The RR protein level declined in dogs with moderate and severe HF, whereas NCX protein did not change with moderate HF but increased with sever HF. These results suggest that the regulatory proteins responsible for Ca(2+) uptake, Ca(2+) release, and Na(+)-Ca(2+) exchange are critically associated with the deterioration of LV function during the progression of HF.

Coordinate downregulation of CaM kinase II and phospholamban accompanies contractile phenotype transition in the hyperthyroid rabbit soleus.

This study investigated the effects of l-thyroxine-induced hyperthyroidism on Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaM kinase II)-mediated sarcoplasmic reticulum (SR) protein phosphorylation, SR Ca(2+) pump (Ca(2+)-ATPase) activity, and contraction duration in slow-twitch soleus muscle of the rabbit. Phosphorylation of Ca(2+)-ATPase and phospholamban (PLN) by endogenous CaM kinase II was found to be significantly lower (30-50%) in soleus of the hyperthyroid compared with euthyroid rabbit. Western blotting analysis revealed higher levels of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) 1 ( approximately 150%) Ca(2+) pump isoform, unaltered levels of SERCA2 Ca(2+) pump isoform, and lower levels of PLN ( approximately 50%) and delta-, beta-, and gamma-CaM kinase II (40 approximately 70%) in soleus of the hyperthyroid rabbit. SR vesicles from hyperthyroid rabbit soleus displayed approximately twofold higher ATP-energized Ca(2+) uptake and Ca(2+)-stimulated ATPase activities compared with that from euthyroid control. The V(max) of Ca(2+) uptake (in nmol Ca(2+).mg SR protein(-1).min(-1): euthyroid, 818 +/- 73; hyperthyroid, 1,649 +/- 90) but not the apparent affinity of the Ca(2+)-ATPase for Ca(2+) (euthyroid, 0.97 +/- 0.02 microM, hyperthyroid, 1.09 +/- 0.04 microM) differed significantly between the two groups. CaM kinase II-mediated stimulation of Ca(2+) uptake by soleus muscle SR was approximately 60% lower in the hyperthyroid compared with euthyroid. Isometric twitch force of soleus measured in situ was significantly greater ( approximately 36%), and the time to peak force and relaxation time were significantly lower ( approximately 30-40%), in the hyperthyroid. These results demonstrate that thyroid hormone-induced transition in contractile properties of the rabbit soleus is associated with coordinate downregulation of the expression and function of PLN and CaM kinase II and selective upregulation of the expression and function of SERCA1, but not SERCA2, isoform of the SR Ca(2+) pump.

Evidence for symplastic involvement in the radial movement of calcium in onion roots.

The pathway of Ca(2+) movement from the soil solution into the stele of the root is not known with certainty despite a considerable body of literature on the subject. Does this ion cross an intact, mature exodermis and endodermis? If so, is its movement through these layers primarily apoplastic or symplastic? These questions were addressed using onion (Allium cepa) adventitious roots lacking laterals. Radioactive Ca(2+) applied to the root tip was not transported to the remainder of the plant, indicating that this ion cannot be supplied to the shoot through this region where the exodermis and endodermis are immature. A more mature zone, in which the endodermal Casparian band was present, delivered 2.67 nmol of Ca(2+) mm(-1) treated root length d(-1) to the transpiration stream, demonstrating that the ion had moved through an intact endodermis. Farther from the root tip, a third zone in which Casparian bands were present in the exodermis as well as the endodermis delivered 0.87 nmol Ca(2+) mm(-1) root length d(-1) to the transpiration stream, proving that the ion had moved through an unbroken exodermis. Compartmental elution analyses indicated that Ca(2+) had not diffused through the Casparian bands of the exodermis, and inhibitor studies using La(3+) and vanadate (VO(4)(3-)) pointed to a major involvement of the symplast in the radial transport of Ca(2+) through the endodermis. It was concluded that in onion roots, the radial movement of Ca(2+) through the exodermis and endodermis is primarily symplastic.

Rotenone-sensitive mitochondrial potential in Phytomonas serpens: electrophoretic Ca 2+ accumulation

Phytomonas sp. are flagellated trypanosomatid plant parasites that cause diseases of economic importance in plantations of coffee, oil palm, cassava and coconuts. Here we investigated Ca 2+ uptake by the vanadate-insensitive compartments using permeabilized Phytomonas serpens promastigotes. This uptake occurs at a rate of 1.13±0.23 nmol Ca 2+ mg protein −1 min −1. It is completely abolished by the H + ionophore FCCP and by valinomycin and nigericin. It is also inhibited by 2 μM ruthenium red, which, at this low concentration, is known to inhibit the mitochondrial calcium uniport. Furthermore, salicylhydroxamic acid (SHAM) and propylgallate, specific inhibitors of the alternative oxidase in plant and parasite mitochondria, are also effective as inhibitors of the Ca 2+ transport. These compounds abolish the membrane potential that is monitored with safranine O. Rotenone, an inhibitor of NADH-CoQ oxidoreductase, can also dissipate 100% of the membrane potential. It is suggested that the mitochondria of P. serpens can be energized via oxidation of NADH in a pathway involving the NADH-CoQ oxidoreductase and the alternative oxidase to regenerate the ubiquinone. The electrochemical H + gradient can be used to promote Ca 2+ uptake by the mitochondria.

Lipid peroxidation and active calcium transport in inside–out vesicles of red blood cells from preeclamptic women

We previously reported that in preeclampsia Ca-ATPase activity diminishes about 50% in red blood cells, myometrium and syncitiotrophoblast plasma membranes. In this work, we measured the active Ca ++ uptake by inside–out vesicles of human red blood cells from preeclamptic and normotensive pregnant women. Active calcium uptake by the vesicles was diminished by 49±3% in the preeclamptic women as compared to the gestational controls (8.06±0.11 nmol Ca ++/mg protein min, gestational controls; 4.08±0.1 nmol Ca ++/mg protein min, preeclamptics). This lowered calcium uptake correlates well with the lowered Ca-ATPase activity found in the red blood cells ghosts of the preeclamptic women (17.05±0.96 nmol Pi/mg protein min, gestational controls; 8.85±0.45 nmol Pi/mg protein min, preeclamptics). The reduced calcium uptake and Ca-ATPase activity of the red cell membranes both appear to be associated with a high level of lipid peroxidation. Thus there is a diminution in the active transport of calcium in the red blood cells of preeclamptic women. If this also occurs in other cell types of the preclamptic women, it could result in an increase in their cytosolic calcium concentration which might be responsible, in part, for some of the symptoms of this disease.

Effect of zooplankton availability on the rates of photosynthesis, and tissue and skeletal growth in the scleractinian coral Stylophora pistillata

This work investigated the effect of light and feeding on tissue composition as well as on rates of photosynthesis and calcification in the zooxanthellae (zoox) scleractinian coral, Stylophora pistillata. Microcolonies were maintained at three different light levels (80, 200, 300 μmol m −2 s −1) and subjected to two feeding regimes (starved and fed) over 9 weeks. Corals were fed both natural plankton and Artemia salina nauplii four times a weeks and samplings were made after 2, 5, and 9 weeks. Results confirmed that feeding enhances coral growth rate and increases both the dark and light calcification rates. These rates were 50–75% higher in fed corals (FC; 60±20 and 200±40 nmol Ca 2+ cm −2 h −1 for dark and light calcification, respectively) compared to control corals (CC; 30±9 and 124±23 nmol Ca 2+ cm −2 h −1). The dark calcification rates, however, were four times lower than the rates of light calcification (independent of trophic status). After 5 weeks, chlorophyll a (chl- a) concentrations were four to seven times higher in fed corals (7–21 μg cm −2) than in control corals (2–5 μg cm −2). The amount of protein was also significantly higher in fed corals (2.11–2.50 mg cm −2) than in control corals (1.08–1.52 mg cm −2). Rates of photosynthesis in fed corals were 2–10 times higher (1.24±0.75 μmol O 2 h −1 cm −2) than those measured in control corals (0.20±0.08 μmol O 2 h −1 cm −2).

Thyroid hormone may induce changes in the concentration of the mitochondrial calcium uniporter

We explored the possibility that the hormone 3,3′,5-tri-iodothyronine can regulate the biosynthesis of the mitochondrial calcium uniporter. To meet this objective experiments on Ca 2+ transport, and binding of the specific inhibitor Ru 360 were carried out in mitochondria isolated from euthyroid, hyperthyroid and hypothyroid rats. It was found that V max for Ca 2+ transport increased from 11.67±0.8 in euthyroid to 14.36±0.44 in hyperthyroid, and decreased in hypothyroid mitochondria to 8.62±0.63 nmol Ca 2+/mg/s. Furthermore, the K i for the specific inhibitor Ru 360, depends on the thyroid status, i.e. 18, 19 and 13 nM for control, hyper- and hypothyroid mitochondria, respectively. In addition, the binding of 103Ru 360 was increased in hyperthyroid and decreased in hypothyroid mitochondria. Scatchard analysis for the binding of 103Ru 360 showed the following values: 28, 40 and 23 pmol/mg for control, hyper- and hypothyroid mitochondria, respectively. The K d for 103Ru 360 was found to be 30.39, 37.03 and 35.71 nM for controls, hyper- and hypothyroid groups, respectively. When hypothyroid rats were treated with thyroid hormone, mitochondrial Ca 2+ transport, as well as 103Ru 360 binding, reached similar values to those found for euthyroid mitochondria.

Compartmental analysis of steady-state diaphragm Ca 2+ kinetics in chronic congestive heart failure

An analytic method based on simulation and modeling of long-term 45 Ca 2+ efflux data was used to estimate steady-state Ca 2+ contents (nmol Ca 2+ g −1 tissue wet wt.) and exchange fluxes (nmol Ca 2+ min −1 g −1 tissue wet wt.) for extracellular and intracellular compartments in in vitro resting diaphragm from congestive heart failure (CHF, n=12) and sham-operated (SHAM, n=10) rats. Left hemidiaphragms were excised from experimental animals, loaded with 45 Ca 2+ for 1 h, and washed out with 45 Ca 2+ -free perfusate for 8 h. Tissue from the right hemidiaphragm was used to assess single-fiber cross-sectional area (CSA) as well as the relative proteolytic activity of Ca 2+-dependent calpain. Kinetic analysis of 45 Ca 2+ efflux data revealed that CHF was associated with increased Ca 2+ contents of extracellular and intracellular compartments as well as increased Ca 2+ exchange fluxes for all compartments. This accounted for the model prediction of a 250% increase in total diaphragm Ca 2+. Furthermore, single-fiber CSA was decreased 12% and proteolytic activity of calpain was increased twofold in CHF diaphragm relative to SHAM. Conclusions: The kinetic data are consistent with the hypothesis that diaphragm Ca 2+ overload in CHF required all intercompartmental Ca 2+ fluxes to increase. The potential relationships among Ca 2+ overload, increased activity of calpain, and wasting of the diaphragm in CHF are discussed.

Nucleolin is a calcium-binding protein.

We have purified a prominent 110-kDa protein (p110) from 1.6 M NaCl extracts of rat liver nuclei that appears to bind Ca2+. p110 was originally identified by prominent blue staining with 'Stains-All' in sodium dodecyl sulfate-polyacrylamide gels and was observed to specifically bind ruthenium red and 45Ca2+ in nitrocellulose blot overlays. In spin-dialysis studies, purified p110 saturably bound approximately 75 nmol Ca2+/mg protein at a concentration of 1 mM total Ca2+ with half-maximal binding observed at 105 microM Ca2+. With purification, p110 became increasingly susceptible to proteolytic (likely autolytic) fragmentation, although most intermediary peptides between 40 and 90 kDa retained "Stains-All", ruthenium red, and 45Ca2+ binding. N-terminal sequencing of intact p110 and a 70-kDa autolytic peptide fragment revealed a strong homology to nucleolin. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/IEF revealed autolysis produced increasingly acidic peptide fragments ranging in apparent pI's from 5.5 for intact p110 to 3.5 for a 40 kDa peptide fragment. Intact p110 and several peptide fragments were immunostained with a highly specific anti-nucleolin antibody, R2D2, thus confirming the identity of this protein with nucleolin. These annexin-like Ca2+-binding characteristics of nucleolin are likely contributed by its highly acidic argyrophilic N-terminus with autolysis apparently resulting in largely selective removal of its basic C-terminal domain. Although the Ca2+-dependent functions of nucleolin are unknown, we discuss the possibility that like the structurally analogous HMG-1, its Ca2+-dependent actions may regulate chromatin structure, possibly during apoptosis.

Interspecies differences in hepatic Ca(2+)-ATPase activity and the effect of cold preservation on porcine liver Ca(2+)-ATPase function.

The accumulation of intracellular calcium ([Ca(2+)](i)) caused by ischemia-reperfusion during liver transplantation has been implicated as a factor leading to primary graft nonfunction. Plasma membrane (PM) and endoplasmic reticulum (ER) Ca(2+)-adenosinetriphosphatases (ATPases) are the primary transporters that maintain [Ca(2+)](i) homeostasis in the liver. We hypothesized that the porcine liver is better than the rat liver as a model for the study of human liver Ca(2+)-ATPase activity. We also hypothesized that cold preservation would depress Ca(2+)-ATPase activity in the porcine liver. Pig and rat livers were harvested, and human liver samples were obtained from surgical resection specimens. All were preserved with University of Wisconsin solution, and porcine livers were also preserved on ice for 2 to 18 hours. Ca(2+)-ATPase activity was measured after incubation with (45)Ca(2+) and adenosine triphosphate in the presence of specific Ca(2+)-ATPase inhibitors. Porcine PM and ER Ca(2+)-ATPase activities were 0.47 +/- 0.03 and 1.57 +/- 0.10 nmol of Ca(2+)/mg of protein/min, respectively. This was not significantly different from human liver, whereas rat liver was significantly greater at 2.60 +/- 0.03 and 9.2 +/- 0.9 nmol of Ca(2+)/mg of protein/min, respectively. We conclude that the Ca(2+)-ATPase activity in the pig liver is equivalent to that of human liver, and thus, the pig liver is a better model than the rat liver. Cold preservation studies showed a significant decrease in porcine hepatic PM Ca(2+)-ATPase activity after 4 hours of storage and near-total inhibition after 12 hours. Porcine hepatic ER Ca(2+)-ATPase activity showed a 45% decrease in activity by 12 hours and a 69% decrease by 18 hours. We conclude that cold ischemia at clinically relevant times depresses PM Ca(2+)-ATPase more than ER Ca(2+)-ATPase activity in pig liver homogenates.

Cardiac ischemia oxidizes regulatory thiols on ryanodine receptors: captopril acts as a reducing agent to improve Ca2+ uptake by ischemic sarcoplasmic reticulum.

We tested the hypothesis that ischemia alters sarcoplasmic reticulum (SR) Ca2+ transport by oxidizing regulatory thiols on ryanodine receptors (RyRs), and that membrane-permeable sulfhydryl-containing angiotensin-converting enzyme (ACE) inhibitors protect against ischemia-induced oxidation and explain in part, the therapeutic actions of captopril. Ca2+ uptake and adenosine triphosphatase (ATPase) activity was measured from SR vesicles isolated from control or ischemic dog and human ventricles and compared with or without sulfhydryl reductants. The rate and amount of Ca2+ uptake was lower for canine ischemic SR compared with control (6.5 +/- 0.2 --> 18.5 +/- 1.1 nmol Ca2+/mg/min and 123.1 +/- 4.7 --> 235.0 +/- 17.3 nmol Ca2+/mg; n = 8 each). Captopril, dithiothreitol (DTT), glutathione (GSH), and L-cysteine increased the rate and amount of Ca2+ uptake by canine and human ischemic SR vesicles by approximately 50%. Reducing agents had no effect on Ca2+- ATPase activity in either canine control or ischemic (approximately 40% less than control) SR. Captopril was as potent as DTT at reversing the oxidation of skeletal and cardiac RyRs induced by reactive disulfides (RDSs) or nitric oxide (NO). In neonatal rat myocytes, RDSs or NO triggered SR Ca2+ release and increased cytosolic Ca2+, an effect reversed by captopril and DTT but not GSH or cysteine. Pretreatment of myocytes with captopril (exposure and then wash) inhibited Ca2+ elevation elicited by RDSs or NO, indicating that captopril is an effective, membrane-permeable intracellular reducing agent. Thus, net SR Ca2+ accumulation is reduced by ischemia in part due to the oxidation of thiols that gate RyRs, an effect reversed by captopril.

Energy-dependent Ca(2+) efflux from the cells of nostoc calcicola Breb: role of modifying factors

Energy-dependent Ca(2+) efflux and its regulation from the diazotrophic cyanobacterium Nostoc calcicola Breb has been investigated. Like Ca(2+) uptake, Ca(2+) efflux pattern also reflected a rapid phase for the first 10 min followed by a slower one lasting up to 1 h with a total of 80 nmol Ca(2+) mg(-1) protein (31% of the Ca(2+) concentration taken in by such cells at 1 h). Ca(2+) efflux kinetics remained hyperbolic with a K(m) of 1.9 mM and V(max) 5.5 nmol mg(-1) protein min(-1). Ca(2+) efflux to a major extent depended on photosynthetic energy generation as the cells facing dark incubation and addition of 3-(3, 4-dichlorophenyl)-1-dimethyl urea (DCMU) to light-grown cells showed significant reduction in Ca(2+) extrusion. The strong inhibition in Ca(2+) efflux by addition of metabolic inhibitors like carbonyl cyanide-p-nitrofluoromethoxylphenyl hydrazone (FCCP) and N,N, -dicyclohexylcarbo-diimide (DCCD) suggested the vital role of membrane potential and ATP hydrolysis in driving this process. Verapamil (Ca(2+) antagonist) had insignificant effect on Ca(2+) efflux, whereas the addition of Calmodulin antagonists like trifluoroperazine, W-7 and compound 48/80 resulted in the enhancement in Ca(2+) efflux over control sets, thus suggesting that this increase may be owing to the additional extrusion of intracellular free calcium that was unable to bind with calmodulin in the presence of these antagonists.http://link.springer-ny. com/link/service/journals/00284/bibs/39n5p254.html</HEA

Synaptic vesicle Ca2+/H+ antiport: dependence on the proton electrochemical gradient

Synaptic vesicles isolated from sheep brain cortex accumulate Ca2+ by a mechanism of secondary active transport associated to the H+-pump activity. The process can be visualized either by measuring Ca2+-induced H+ release or ΔpH-dependent Ca2+ accumulation. We observed that the amount of Ca2+ taken up by the vesicles increases with the magnitude of the ΔpH across the membrane, particularly at Ca2+ concentrations (∼500 μM) found optimal for the antiporter activity. Similarly, H+ release induced by Ca2+ increased with the magnitude of ΔpH. However, above 60% ΔpH (high H+-pump activity), the net H+ release from the vesicles decreased as the pump-mediated H+ influx exceeded the Ca2+-induced H+ efflux. We also observed that the Ca2+/H+ antiport activity depends, essentially, on the ΔpH component of the electrochemical gradient (∼3 nmol Ca2+ taken up/mg protein), although the Δϕ component may also support some Ca2+ accumulation by the vesicles (∼1 nmol/mg protein) in the absence of ΔpH. Both Ca2+-induced H+ release and ΔpH-dependent Ca2+ uptake could be driven by an artificially imposed proton motive force. Under normal conditions (H+ pump-induced ΔpH), the electrochemical gradient dependence of Ca2+ uptake by the vesicles was checked by inhibition of the process with specific inhibitors (bafilomycin A1, ergocryptin, folymicin, DCCD) of the H+-pump activity. These results indicate that synaptic vesicles Ca2+/H+ antiport is indirectly linked to ATP hydrolysis and it is essentially dependent on the chemical component (ΔpH) of the electrochemical gradient generated by the H+-pump activity.

Beneficial effects of propionyl L-carnitine on sarcolemmal changes in congestive heart failure due to myocardial infarction.

Earlier studies have revealed sarcolemmal (SL) defects in congestive heart failure due to myocardial infarction; however, the mechanisms of SL changes in the failing heart are poorly understood. Since congestive heart failure is associated with various metabolic abnormalities including a deficiency of carnitine, we examined the effects of propionyl L-carnitine, a carnitine derivative, in animals with congestive heart failure. For this purpose, heart failure in rats was induced by occluding the coronary artery and 3 weeks later the animals were treated with 100 mg/kg (i.p. daily) propionyl L-carnitine for 4 weeks. The sham control group received saline injections. The animals were assessed for their left ventricular function. SL membranes were examined for Na(+)-K+ ATPase, Na(+)-Ca2+ exchange and adenylate cyclase activities. A marked improvement in the attenuated left ventricular function of the experimental animals was seen upon treatment with propionyl L-carnitine. The SL adenylyl cyclase activities in control, untreated failing hearts and treated failing hearts were 590 +/- 36, 190 +/- 22 and 320 +/- 21 pmol cAMP/mg/10 min, whereas the SL Na(+)-K+ ATPase activities were 35.7 +/- 2.8, 22.5 +/- 2.4 and 30.1 +/- 2.8 mumol Pi/mg/h, respectively. Furthermore, the SL Na(+)-dependent Ca(2+)-uptake activity, which decreased in the failing hearts (4.6 +/- 0.4 vs. 9.3 +/- 0.7 nmol Ca2+/mg/2 s for control), was improved (6.8 +/- 0.5 nmol Ca2+/mg/2 s) significantly following treatment with propionyl L-carnitine. These results indicate that metabolic therapy with propionyl L-carnitine may attenuate defects in the SL membrane and thus may improve heart function in congestive heart failure due to myocardial infarction.

Identification of a Ca2+/H+ antiport in the plant chloroplast thylakoid membrane

To assess the availability of Ca2+ in the lumen of the thylakoid membrane that is required to support the assembly of the oxygen-evolving complex of photosystem II, we have investigated the mechanism of 45Ca2+ transport into the lumen of pea (Pisum sativum) thylakoid membranes using silicone-oil centrifugation. Trans-thylakoid Ca2+ transport is dependent on light or, in the dark, on exogenously added ATP. Both light and ATP hydrolysis are coupled to Ca2+ transport through the formation of a transthylakoid pH gradient. The H+-transporting ionophores nigericin/K+ and carbonyl cyanide 3-chlorophenylhydrazone inhibit the transport of Ca2+. Thylakoid membranes are capable of accumulating up to 30 nmol Ca2+ mg-1 chlorophyll from external concentrations of 15 μM over the course of a 15-min reaction. These results are consistent with the presence of an active Ca2+/H+ antiport in the thylakoid membrane. Ca2+ transport across the thylakoid membrane has significant implications for chloroplast and plant Ca2+ homeostasis. We propose a model of chloroplast Ca2+ regulation whereby the activity of the Ca2+/H+ antiporter facilitates the light-dependent uptake of Ca2+ by chloroplasts and reduces stromal Ca2+ levels.

Direct evidence of Na+/Ca2+ exchange in squid rhabdomeric membranes.

Na+/Ca2+ exchange has been investigated in squid (Loligo pealei) rhabdomeric membranes. Ca2+-containing vesicles have been prepared from purified rhabdomeric membranes by extrusion through polycarbonate filters of 1-micrometer pore size. After removal of external Ca2+, up to 90% of the entrapped Ca2+ could be specifically released by the addition of Na+; this finding indicates that most of the vesicles contained Na+/Ca2+ exchanger. The Na+-induced Ca2+ efflux had a half-maximum value (K1/2) of approximately 44 mM and a Hill coefficient of approximately 1.7. The maximal Na+-induced Ca2+ efflux was approximately 0.6 nmol Ca2+. s-1. mg protein-1. Similar Na+-induced Ca2+ effluxes were measured if K+ was replaced with Li+ or Cs+. Vesicles loaded with Ca2+ by Na+/Ca2+ exchange also released this Ca2+ by Na+/Ca2+ exchange, suggesting that Na+/Ca2+ exchange operated in both forward and reverse modes. Limited proteolysis by trypsin resulted in a rate of Ca2+ efflux enhanced by approximately fivefold when efflux was activated with 95 mM NaCl. For vesicles subjected to limited proteolysis by trypsin, Na+/Ca2+ exchange was characterized by a K1/2 of approximately 25 mM and a Hill coefficient of 1.6. For these vesicles, the maximal Na+-induced Ca2+ efflux was about twice as great as in control vesicles. We conclude that Na+/Ca2+ exchange proteins localized in rhabdomeric membranes mediate Ca2+ extrusion in squid photoreceptors.

Salicylate-induced kidney mitochondrial permeability transition is prevented by cyclosporin A

The effect of salicylate, the active metabolite of aspirin (acetyl salicylic acid) in the presence of Ca 2+ and phosphate on mitochondrial permeability transition (MPT) was studied. MPT is often associated with opening of a Ca 2+-induced pore. The opening of this pore leads to swelling, loss of mitochondrial membrane potential and release of accumulated Ca 2+. In freshly isolated rat kidney mitochondria, salicylate (400 μM) in the presence of 20 nmol Ca 2+/mg protein and 0.1 mM phosphate induced swelling, loss of mitochondrial membrane potential and release of accumulated Ca 2+. All these changes were eliminated when cyclosporin A (1 μM), (a pore inhibitory agent) was included in the incubation medium. Unlike salicylate, unhydrolyzed aspirin (400 μM) induced these changes slightly. We concluded that salicylate acts as an activator of Ca 2+ and phosphate in promoting the opening of kidney inner mitochondrial membrane pore. As a result a great consideration should be given to its toxicological effect.

Large-Scale Expression of Recombinant Cardiac Sodium–Calcium Exchange in Insect Larvae

Recombinant bovine cardiac sodium–calcium exchange (NCX1) in a baculovirus construct was used to infect cabbage looper larvae (Trichoplusia ni). Infected larvae were homogenized and larvae membrane vesicles were purified. Western blot analysis indicated the presence of recombinant NCX1 protein in vesicles from infected larvae but not in controls. Vesicles from infected larvae expressed high levels of NCX1 activity (1.7 nmol Ca2+/mg protein/s) while vesicles from control larvae had no activity. NCX1 in larvae vesicles was bidirectional. Kinetic analysis yielded aVmaxof 3.6 nmol Ca2+/mg protein/s and aKmfor Ca of 4.2 μM. NCX1 activity was inhibited by the exchange inhibitory peptide with an IC50of 4 μM. These data demonstrate a novel and efficient method for the expression of large amounts of active recombinant NCX1 protein that has general application for expression and analysis of recombinant membrane proteins.

Changes in calcium uptake rate by rat cardiac mitochondria during postnatal development.

Ca2+ uptake, transmembrane electrical potential (Deltapsim) and oxygen consumption were measured in isolated ventricular mitochondria of rats from 3 days to 5 months of age. Estimated values of ruthenium red-sensitive, succinate-supported maximal rate of Ca2+ uptake (Vmax, expressed as nmol Ca2+/min/mg protein) were higher in neonates and gradually fell during postnatal development (from 435+/-24 at 3-6 days, to 156+/-10 in adults,P<0.001), whereas K0.5 values (approximately 10 microM were not significantly affected by age. Under similar conditions, mitochondria from adults (5 months old) and neonates (4-6 days old) showed comparable state 4 (succinate and alpha-ketoglutarate as substrates) and state 3ADP (alpha-ketoglutarate-supported) respiration rates, as well as Deltapsim values (approximately-150 mV). Respiration-independent Deltapsim and Ca2+ uptake, supported by valinomycin-induced K+ efflux were also investigated at these ages. A transient Deltapsim (approximately -30 mV) was evoked by valinomycin in both neonatal and adult mitochondria. Respiration-independent Ca2+ uptake was also transient, but its initial rate was significantly higher in neonates than in adults (49. 4+/-10.0v 28.0+/-5.7 mmol Ca2+/min/mg protein,P<0.01). These results indicate that Ca2+ uptake capacity of rat cardiac mitochondria is remarkably high just after birth and declines over the first weeks of postnatal life, without change in apparent affinity of the transporter. Increased mitochondrial Ca2+ uptake rate in neonates appears to be related to the uniporter itself, rather than to modification of the driving force of the transport.