Inhibition of ethylene production by cobaltous ion

The effect of Co²⁺ on ethylene production by mung bean (Phaseolus aureus Roxb.) and by apple tissues was studied. Co²⁺, depending on concentrations applied, effectively inhibited ethylene production by both tissues. It also strongly inhibited the ethylene production induced by IAA, kinetin, IAA plus kinetin, Ca²⁺, kinetin plus Ca²⁺, or Cu²⁺ treatments in mung bean hypocotyl segments. While Co²⁺ greatly inhibited ethylene production, it had little effect on the respiration of apple tissue, indicating that Co²⁺ does not exert its inhibitory effect as a general metabolic inhibitor. Ni²⁺, which belongs to the same group as Co²⁺ in the periodic table, also markedly curtailed both the basal and the induced ethylene production by apple and mung bean hypocotyl tissues.     

Participation of phenolic acids of microbial origin in the dysfunction of mitochondria in sepsis

The role of low-molecular-weight phenolic acids of microbial origin in the mitochondrial dysfunction observed in sepsis has been studied. It was shown that microbial phenolic acids formed during fermentation of aromatic amino acids and polyphenols have an effect on mitochondrial functions, whose magnitude depends on the structure of a particular phenolic acid. The anaerobic metabolites cinnamic and benzoic acids and, to a lesser extent, phenylpropionic and phenylacetic acids at concentrations of 0.02–0.1 mM inhibited the NAD-dependent respiration, decreased the Ca²⁺-retention capacity of mitochondria, and oxidized the thiol groups. Their effects were partially abolished by menadione and dithiothreitol. Hydroxylated phenolic acids, 2,4-dihydroxybenzoic, 2,3-dihydroxyphenylpropionic, and other phenolic acids formed in aerobic metabolism of bacteria, when used at the same concentrations, did not affect these processes. During the catabolism of phenolic acids by clinically important bacteria, these compounds undergo anaerobic interconversions. The data obtained suggest that they contribute to the mitochondrial dysfunction in sepsis, and this contribution increases under hypoxic conditions.     

Cytokinin modification of mitochondrial function

6-Benzylaminopurine, 6-(Δ²-isopentenylamino)purine, 6-furfurylaminopurine, rotenone, and antimycin A inhibited oxidation of NADH by mitochondrial sonicates or submitochondrial particles (but not by intact mitochondria) from pea (Pisum sativum L., cult. Alaska) stems and mung bean (Vigna radiata L. Wilczak) hypocotyls. The above purine cytokinins can interfere with electron transport from NADH to the cytochrome system in the inner mitochondrial membrane. Adenine did not inhibit oxidation by sonicated mitochondria, and zeatin was almost ineffective. Zeatin scarcely inhibited state 3 malate respiration by intact mitochondria, but the O-formyl and O-n-propionyl esters of zeatin and the O-acetyl ester of 2-chlorozeatin were more active. Perhaps zeatin is ineffective because it does not get into the inner membranes of the isolated mitochondria, whereas the esters and other cytokinins mentioned above do. N-4-(2-chloropyridyl)-N′-Phenylurea, which has cytokinin-like effects on plant growth and development, inhibited NADH oxidation by sonicated mitochondria. It also inhibited malate, succinate, and NADH oxidation by intact mitochondria; in contrast, the latter two oxidations were not decreased by purine cytokinins.     

Studies on energy status and mitochondrial respiration during growth and senescence of mung bean cotyledons

Several characteristics of mitochondrial respiration and energy status have been studied during growth and senescence of mung bean ( Phaseolus radiatus L.) cotyledons. The results showed that mitochondrial oxygen consumption, respiratory control, ADP:O ratios, and energy charge changed in the cotyledons during germination and growth of the seedlings. The respiration rate of intact cotyledons approximately reflected the trend of the oxidative activities of the isolated mitochondria. An increase was observed in both whole cotyledon respiration and mitochondrial oxygen uptake at the onset of senescence of mung bean cotyledons (day 3 after germination), which thereafter declined gradually. The capacity and activity of the alternative pathway increased markedly in mitochondria isolated from senescent cotyledons. After the onset of senescence, the mung bean cotyledon mitochondria exhibited a decrease both in the respiratory control ratios and ADP:O ratios, and the cotyledons exhibited a gradual decline in energy charge. All these results showed an irreversible deterioration of energy conservation in mung bean cotyledons. The role(s) of the alternative pathway in senescent mung bean cotyledons is discussed.     

Brain mitochondria from rats treated with sulforaphane are resistant to redox-regulated permeability transition

Oxidative stress promotes Ca²⁺-dependent opening of the mitochondrial inner membrane permeability transition pore (PTP), causing bioenergetic failure and subsequent cell death in many paradigms, including those related to acute brain injury. One approach to pre-conditioning against oxidative stress is pharmacologic activation of the Nrf2/ARE pathway of antioxidant gene expression by agents such as sulforaphane (SFP). This study tested the hypothesis that administration of SFP to normal rats increases resistance of isolated brain mitochondria to redox-sensitive PTP opening. SFP or DMSO vehicle was administered intraperitoneally to adult male rats at 10 mg/kg 40 h prior to isolation of non-synaptic brain mitochondria. Mitochondria were suspended in medium containing a respiratory substrate and were exposed to an addition of Ca²⁺ below the threshold for PTP opening. Subsequent addition of tert-butyl hydroperoxide (tBOOH) resulted in a cyclosporin A-inhibitable release of accumulated Ca²⁺ into the medium, as monitored by an increase in fluorescence of Calcium Green 5N within the medium, and was preceded by a decrease in the autofluorescence of mitochondrial NAD(P)H. SFP treatment significantly reduced the rate of tBOOH-induced Ca²⁺ release but did not affect NAD(P)H oxidation or inhibit PTP opening induced by the addition of phenylarsine oxide, a direct sulfhydryl oxidizing agent. SFP treatment had no effect on respiration by brain mitochondria and had no effect on PTP opening or respiration when added directly to isolated mitochondria. We conclude that SFP confers resistance of brain mitochondria to redox-regulated PTP opening, which could contribute to neuroprotection observed with SFP.     

Ca 2+ transport activity in mitochondria from some plant tissues

Mitochondria isolated from some 14 different higher plants and fungi were examined for their capacity to carry out respiration-dependent accumulation of Ca²⁺. Additions of Ca²⁺ give little or no stimulation of state 4 respiration of plant mitochondria, although the added Ca²⁺ was largely accumulated. Accumulation of Ca²⁺ required phosphate and, in most cases, was stimulated by Mg²⁺ and ADP or ATP. Ca²⁺ uptake was abolished by respiratory inhibitors and uncoupling agents. The ratio of Ca²⁺ ions taken up per pair of electrons per energy-conserving site was normal at about 2.0 for mitochondria from sweet potato and white potato; mitochondria from other plants showed somewhat lower ratios. Accumulated Ca²⁺ was only very slowly released from previously loaded plant mitochondria. Respiration-inhibited sweet potato mitochondria show both high-affinity and low-affinity Ca²⁺ binding sites sensitive to uncouplers, La³⁺, and ruthenium red and thus resemble animal mitochondria. Most other plant mitochondria lack high affinity sites. In general, mitochondria from sweet potato and white potato tubers resemble those from animal tissues, but mitochondria from carrots, beets, turnips, onions, cabbage, artichokes, cauliflower, avocados, mung bean and corn seedlings, and mushrooms show rather low affinity and activity in accumulation of Ca²⁺, probably due to lack of a specific Ca²⁺ carrier.     

Effects of Fluoride on Mitochondrial Activity in Higher Plants

The effects of fluoride on respiration of plant tissue and mitochondria were investigated. Fumigation of young soybean plants (Glycine max Merr. cv. Hawkeye) with 9–12 μg × m^?3 HF caused a stimulation of respiration at about 2 days of treatment followed by inhibition 2 days later. Mitochondria isolated from the stimulated tissue had higher respiration rates, greater ATPase activity, and lower P/O ratios, while in mitochondria from inhibited tissue, all three were reduced. Treatment of etiolated soybean hypocotyl sections in Hoagland's solution containing KF for 3 to 10 h only resulted in inhibition of respiration. Mitochondria isolated from this tissue elicited increased respiration rates with malate as substrate and inhibited respiration with succinate. With both substrates respiratory control and ADP/O ratios were decreased. Direct treatment of mitochondria from the etiolated soybean hypocotyl tissue with fluoride resulted in inhibition of state 3 respiration and lower ADP/O ratios with the substrates succinate, malate, and NADH. Fluoride was also found to increase the amount of osmotically induced swelling and cause a more rapid leakage of protein with mitochondria isolated from etiolated corn shoots (Zea mays L. cv. Golden Cross Bantam). The results are discussed with respect to possible effects of fluoride on mitochondrial membranes.     

Mitochondrial Remodeling in Endothelial Cells under Cyclic Stretch is Independent of Drp1 Activation

Mitochondria in endothelial cells remodel morphologically when supraphysiological cyclic stretch is exerted on the cells. During remodeling, mitochondria become shorter, but how they do so remains elusive. Drp1 is a regulator of mitochondrial morphologies. It shortens mitochondria by shifting the balance from mitochondrial fusion to fission. In this study, we hypothesized that Drp1 activation is involved in mitochondrial remodeling under supraphysiological cyclic stretch. To verify the involvement of Drp1, its activation was first quantified with Western blotting, but Drp1 was not significantly activated in endothelial cells under supraphysiological cyclic stretch. Next, Drp1 activation was inhibited with Mdivi-1, but this did not inhibit mitochondrial remodeling. Intracellular Ca²⁺ increase activates Drp1 through calcineurin. First, we inhibited the intracellular Ca²⁺ increase with Gd³⁺ and thapsigargin, but this did not inhibit mitochondrial remodeling. Next, we inhibited calcineurin with cyclosporin A, but this also did not inhibit mitochondrial remodeling. These results indicate that mitochondrial remodeling under supraphysiological cyclic stretch is independent of Drp1 activation. In endothelial cells under supraphysiological cyclic stretch, reactive oxygen species (ROS) are generated. Mitochondrial morphologies are remodeled by ROS generation. When ROS was eliminated with N-acetyl-L-cysteine, mitochondrial remodeling was inhibited. Furthermore, when the polymerization of the actin cytoskeleton was inhibited with cytochalasin D, mitochondrial remodeling was also inhibited. These results suggest that ROS and actin cytoskeleton are rather involved in mitochondrial remodeling. In conclusion, the present results suggest that mitochondrial remodeling in endothelial cells under supraphysiological cyclic stretch is induced by ROS in association with actin cytoskeleton rather than through Drp1 activation.     

The effect of DS16570511, a new inhibitor of mitochondrial calcium uniporter,on calcium homeostasis,metabolism, and functional state of cultured cortical neurons and isolated brain mitochondria

BackgroundDisorders of mitochondrial Ca²⁺ homeostasis play a key role in the glutamate excitotoxicity of brain neurons. DS16570511 (DS) is a new penetrating inhibitor of mitochondrial Ca²⁺ uniporter complex (MCUC). The paper examines the effects of DS on the cultivated cortical neurons and isolated mitochondria of the rat brain.MethodsThe functions of neurons and mitochondria were examined using fluorescence microscopy, XF24 microplate-based сell respirometry, ion-selective microelectrodes, spectrophotometry, and polarographic technique.ResultsAt the doses of 30 and 45 μM, DS reliably slowed down the onset of glutamate-induced delayed calcium deregulation of neurons and suppressed their death. 30 μM DS caused hyperpolarization of mitochondria of resting neurons, and 45 μM DS temporarily depolarized neuronal mitochondria. It was also demonstrated that 30–60 μM DS stimulated cellular respiration. DS was shown to suppress Ca²⁺ uptake by isolated brain mitochondria. In addition, DS inhibited ADP-stimulated mitochondrial respiration and ADP-induced decrease in the mitochondrial membrane potential. It was found that DS inhibited the activity of complex II of the respiratory chain. In the presence of Ca²⁺, high DS concentrations caused a collapse of the mitochondrial membrane potential.ConclusionsThe data obtained indicate that, in addition to the inhibition of MCUC, DS affects the main energy-transducing functions of mitochondria.General significanceThe using DS as a tool for studying MCUC and its functional role in neuronal cells should be done with care, bearing in mind multiple effects of DS, a proper evaluation of which would require multivariate analysis.     

Spegazzinine,a new inhibitor of mitochondrial oxidative phosphorylation

The effects of spegazzinine, a dihydroindole alkaloid, on mitochondrial oxidative phosphorylation were studied.Spegazzinine inhibited coupled respiration and phosphorylation in rat liver mitochondria. The I₅₀ was 120 μM. Uncouplers released the inhibition of coupled respiration. Arsenate-stimulated mitochondrial respiration was partially inhibited by spegazzinine. The stimulation of mitochondrial respiration by Ca²⁺ and the proton ejection associated with the ATP-dependent Ca²⁺ uptake were not affected by the alkaloid.Oxidative phosphorylation and the P_i-ATP exchange reaction of phosphorylating beef heart submitochondrial particles were strongly inhibited by spegazzinine (I₅₀, 50 μM) while the ATP-dependent reactions, reduction of NAD⁺ by succinate and the pyridine nucleotides transhydrogenase were less sensitive (I₅₀, 125 μM). Oxygen uptake by submitochondrial particles was not affected.The 2,4-dinitrophenol-stimulated ATPase activity of rat liver mitochondria was not affected by 300 μM spegazzinine, a concentration of alkaloid that completely inhibited phosphorylation. However, higher concentrations of spegazzinine did partially inhibit it. The ATPase activities of submitochondrial particles, insoluble and soluble ATPases were also partially inhibited by high concentrations of spegazzinine.The inhibitory properties of spegazzinine on energy transfer reactions are compared with those of oligomycin, aurovertin and dicyclohexylcarbodiimide. It is concluded that spegazzinine effects are very similar to the effects of aurovertin and that its site of action may be the same or near the site of aurovertin.     

Helminthosporium maydis T Toxin Decreased Calcium Transport into Mitochondria of Susceptible Corn

The effects of purified Helminthosporium maydis T (HmT) toxin on active Ca²⁺ transport into isolated mitochondria and microsomal vesicles were compared for a susceptible (T) and a resistant (N) strain of corn (Zea mays). ATP, malate, NADH, or succinate could drive ⁴⁵Ca²⁺ transport into mitochondria of corn roots. Ca²⁺ uptake was dependent on the proton electrochemical gradient generated by the redox substrates or the reversible ATP synthetase, as oligomycin inhibited ATP-driven Ca²⁺ uptake while KCN inhibited transport driven by the redox substrates. Purified native HmT toxin completely inhibited Ca²⁺ transport into T mitochondria at 5 to 10 nanograms per milliliter while transport into N mitochondria was decreased slightly by 100 nanograms per milliliter toxin. Malate-driven Ca²⁺ transport in T mitochondria was frequently more inhibited by 5 nanograms per milliliter toxin than succinate or ATP-driven Ca²⁺ uptake. However, ATP-dependent Ca²⁺ uptake into microsomal vesicles from either N or T corn was not inhibited by 100 nanograms per milliliter toxin. Similarly, toxin had no effect on proton gradient formation ([¹⁴C]methylamine accumulation) in microsomal vesicles. These results show that mitochondrial and not microsomal membrane is a primary site of HmT toxin action. HmT toxin may inhibit formation of or dissipate the electrochemical proton gradient generated by substrate-driven electron transport or the mitochondrial ATPase, after interacting with a component(s) of the mitochondrial membrane in susceptible corn.     

Inhibition of the mitochondrial calcium uniporter by antibodies against a 40-kDa glycorproteinT

Polyclonal rabbit antibodies against a Ca²⁺-binding mitochondrial glycoprotein were found to inhibit the uniporter-mediated transport of Ca²⁺ in mitoplasts prepared from rat liver mitochondria. Spermine, a modulator of the uniporter, decreased the inhibition. This glycoprotein ofM _r 40,000, isolated from beef heart mitochondria and earlier shown to form Ca²⁺-conducting channels in black-lipid membranes, thus is a good candidate for being a component of the uniporter. Antibody-IgG was found to specifically bind to mitochondria in human fibroblasts.     

The effect of phenolic compounds on the activity of respiratory chain enzymes and on respiration and phosphorylation activities of potato tuber mitochondria

The effect of derivatives of benzoic and cinnamic acids, quereetin,p-benzoquinone, and 2,5-dimethylbenzoquinone on oxygen consumption mitoehondrial suspensions and on the activity of some respiratory chain enzymes was studied. Benzoquinone and 2,5-dimethylbenzoquinone highly significantly inhibited the respiration and phosphorylation rates and malate- and succinate dehydrogenase activities. Chlorogenic acid, similarly as the quinones, very significantly inhibited the activities of the studied dehydrogenases but did not affect cytochrome oxidase. Oxygen consumption by intact mitochondria was not inhibited, only the oxidativo phosphorylation was significantly uncoupled. Quereetin significantly enhanced dehydrogenase activities and completely inhibited cytochrome oxidase activity. The respiration and phosphorylation activities of the mitochondria were significantly inhibited by quereetin. The effect of the other phenolic compounds studied on respiration and phosphorylation activities was not significant. Succinate dehydrogenase activity was the most affected enzyme among the respiratory chain enzymes. It was significantly inhibited by all the above phenolic compounds at 1^-4M or 5 10^-5M concentrations with the exception of gallic acid.     

Mitochondrial Generation of Oxygen Radicals During Reoxygenation of Ischemic Tissues

Ischemia and reperfusion causes severe mitochondrial damage, including swelling and deposits of hyd-roxyapatite crystals in the mitochondrial matrix. These crystals are indicative of a massive influx of Ca²⁺ into the mitochondrial matrix occurring during reoxygenation. We have observed that mitochondria isolated from rat hearts after 90 minutes of anoxia followed by reoxygenation, show a specific inhibition in the electron transport chain between NADH dehydrogenase and ubiquinone in addition to becoming uncoupled (unable to generate ATP). This inhibition is associated with an increased H₂O₂ formation at the NADH dehydrogenase level in the presence of NADH dependent substrates. Control rat mitochondria exposed for 15 minutes to high Ca²⁺ (200 nmol/mg protein) also become uncoupled and electron transport inhibited between NADH dehydrogenase and ubiquinone. a lesion similar to that observed in post-ischem-ic mitochondria. This Ca²⁺ -dependent effect is time dependent and may be partially prevented by albumin, suggesting that it may be due to phospholipase A₂ activation. releasing fatty acids, leading to both inhibition of electron transport and uncoupling. Addition of arachidonic or linoleic acids to control rat heart mitochondria, inhibits electron transport between Complex I and III. These results are consistent with the following hypothesis: during ischemia, the intracellular energy content drops severely, affecting the cytoplasic concentration of ions such as Na⁺ and Ca²⁺. Upon reoxygenation, the mitochondrion is the only organelle capable of eliminating the excess cytoplasmic Ca²⁺ through an electrogenic process requiring oxygen (the low ATP concentration makes other ATP-dependent Ca^?' lransport systems non-operational). Ca²⁺-overload of mitochondria activates phospholipase A₂ releasing free fatty acids, leading to uncoupling and inhibition of the interactions between Complex I and III of the respiratory chain. As a consequence, the NADH-dehydrogenase becomes highly reduced, and transfers electrons directly to oxygen generating O₂.     

A Comparison of Mitochondria Isolated from Male-Sterile and Nonserile Cytoplasm Etiolated Corn Seedlings

Toxins from Helminthosporium maydis race T and Phyllosticta maydis have been found to affect the functional processes of corn mitochondria isolated from Texas male-sterile (T) cytoplasm, but not of mitochondria isolated from nonsterile (N) cytoplasm. The effects of chemicals known to induce responses similar to those of the toxin were compared on mitochondria isolated from T and N cytoplasm inbreds (W64A, Zea mays L.). Valinomycin, gramicidin, and decenylsuccinic acid (DSA) each caused more swelling (measured by transmission changes in %) of N mitochondria than of T mitochondira. The stimulation of exogenous NADH oxidation was the same for N and T mitochondria in the valinomycin, DSA, and Ca²⁺ plus phosphate treatments, was greater for T mitochondria than for N mitochondria in the gramicidin and DNP treatments, and was greater for N mitochondrai than for T mitochondira in the Ca⁺² minus phosphate treatment. Sodium azide inhibited NADH oxidation equally for N and T mitochondria. In addition, N and T mitochondria had similar respiration rates for various substrates and equal efficiencies of oxidative phosphorylation. In contrast to the specificity of toxins for T mitochondria, none of the treatment effects were specific for N or T mitochondria. The results indicate that mitochondria isolated from N and T cytoplasm generally respond similarly to various conditions, but that there can be quantitative differences in the response. The extent to which these differences represent cytoplasmically controlled modification of mitochondrial physiology or structure is not known.     

The effect of phosphoenolypyruvate on calcium transport by mitochondria

Phosphoenolpyruvate was found to inhibit net uptake of Ca²⁺ by rat heart and liver mitochondria. The main action of phosphoenolpyruvate is to increase the rate of efflux of mitochondrial Ca²⁺. The effect of phosphoenolpyruvate on mitochondrial Ca²⁺ transport is antagonized by ATP and by atractylate and is observed when mitochondria are respiring in the presence of NAD-linked subtrates such as glutamate and pyruvate plus malate. In liver mitochondria phosphoenolpyruvate is also effective in the presence of succinate but not when rotenone is added. Glycolytic intermdiates other than phosphoenolpyruvate had little effect on mitochondrial Ca²⁺ transport.     

Calcium- and ADP-Magnesium-induced respiratory uncoupling in isolated cardiac mitochondria: Influence of cycolsporin A

This study was designed to determine the effect of calcium and ADP-Mg on the oxidative phosphorylation in isolated cardiac mitochondria. The influence of cyclosporin A was also evaluated. The mitochondria were extracted from rat ventricles. Their oxidative phosphorylations were determined in two respiration media with different free Ca²⁺ concentrations. Respiration was determined with palmitoylcarnitine and either ADP- or ADP-Mg. With elevated free Ca²⁺concentrations and ADP-Mg, the transition state III to state IV respiration did not occurred. The ADP:O ratio was reduced. The phenomenon was not observed in the other experimental conditions (low free Ca²⁺ concentration with either ADP^- or ADP-Mg or elevated free Ca²⁺ concentration with ADP^-). Uncoupling was allied with a constant AMP production, which maintained an elevated ADP level in the respiration medium and prevented the return to state IV respiration. It was also observed in a respiration medium devoid of free Ca²⁺ when the mitochondria were pre-loaded with Ca²⁺. Uncoupling was inhibited by cyclosporin A. Furthermore, the Krebs cycle intermediates released from¹⁴C-palmitoylcarnitine oxidation revealed that succinate was increased by elevated free Ca²⁺ and ADP-Mg. Succinate is a FAD-linked substrate with low respiration efficiency. Its accumulation could account for the decreased ADP:O ratio. The Ca²⁺- and ADP-Mg-induced uncoupling might be partly responsible for the mechanical abnormalities observed during low-flow ischemia. (Mol Cell Biochem 000: 000-000, 1999)     

Mechanisms of antioxidant activities of quercetin and catechin

Abstract

The seleno-organic compound ebselen mimics the glutathione-dependent, hydroperoxide reducing activity of glutathione peroxidase. The activity of glutathione peroxidase determines the rate of hydroperoxide-induced Ca²⁺ release from mitochondria. Ebselen stimulates Ca²⁺ release from mitochondria, accelerates mitochondrial respiration and uncoupling, and induces mitochondrial swelling, indicating a deterioration of mitochondrial function. These manifestations are abolished by cyclo-sporine A, a potent inhibitor of the mitochondrial permeability transition. However, when ebselen-induced Ca²⁺ cycling is prevented with ruthenium red, an inhibitor of the Ca²⁺ uniporter, or by chelation of extramitochondrial Ca²⁺ by EGTA, no detectable elevation of swelling or uncoupling is observed. The release of Ca²⁺ from mitochondria is delayed in the absence of rotenone, i.e. when pyridine nucleotides are maintained in the reduced state due to succinate-driven reversed electron flow. We suggest that ebselen induces Ca²⁺ release from intact mitochondria via an NAD⁺ hydrolysis-dependent mechanism.     

Effect of phosphoenolpyruvate and oxaloacetate on ca uptake by isolated mung bean mitochondria

Phosphoenolpyruvate partially inhibits the accumulation of Ca²⁺ in isolated mung bean (Phaseolus aureus Roxb.) mitochondria. Succinate-supported Ca²⁺ uptake is twice as sensitive to phosphoenolpyruvate inhibition as is NADH- or malate/pyruvate-supported Ca²⁺ uptake. Pyruvate, atractylate, and ATP, but not ITP, reverse the phosphoenolpyruvate-induced inhibition. Oxaloacetic acid inhibits succinate-supported Ca²⁺ uptake completely while partially inhibiting NADH-supported Ca²⁺ uptake. The oxaloacetate inhibition of NADH-supported Ca²⁺ uptake is greater than that produced by phosphoenolpyruvate. It is suggested that inhibition of Ca²⁺ uptake is due to the conversion of phosphoenolpyruvate into oxaloacetate via phosphoenolpyruvate carboxykinase, with oxaloacetate responsible for the actual inhibition of Ca²⁺ uptake.