Inhibitory Properties of Ruthenium Amine Complexes on Mitochondrial Calcium Uptake

The recent finding that the inhibition of Ca²⁺-stimulated respiration by ruthenium red is mainlydue to a binuclear ruthenium complex (Ru₃₆₀) present in the commercial samples of the classicalinhibitor ruthenium red (Ying et. al., 1991), showed that this complex is the more potent andspecific inhibitor of the mitochondrial calcium uniporter. This work was aimed to provideinsights into the mechanism by which Ru₃₆₀ and other ruthenium-related compounds inhibitscalcium uptake. Ruthenium red and a synthesized analog (Rrphen) were compared with Ru₃₆₀.The inhibition by this binuclear complex was noncompetitive, with a K _i of 9.89 nM. Thenumber of specific binding sites for Ru₃₆₀ was 6.2 pmol/mg protein. Ruthenium red and Ru₃₆₀were mutually exclusive inhibitors. Bound La³⁺ was not displaced by Ru₃₆₀. Rrphen was theleast effective for inhibiting calcium uptake. The results support the notion of a specific bindingsite in the uniporter for the polycationic complexes and a negative charged region from thephospholipids in the membrane, closely associated with the uniporter inhibitor-binding site.     

Mitochondrial Calcium Uptake Modulates Synaptic Vesicle Endocytosis in Central Nerve Terminals

Presynaptic calcium influx triggers synaptic vesicle (SV) exocytosis and modulates subsequent SV endocytosis. A number of calcium clearance mechanisms are present in central nerve terminals that regulate intracellular free calcium levels both during and after stimulation. During action potential stimulation, mitochondria rapidly accumulate presynaptic calcium via the mitochondrial calcium uniporter (MCU). The role of mitochondrial calcium uptake in modulating SV recycling has been debated extensively, but a definitive conclusion has not been achieved. To directly address this question, we manipulated the expression of the MCU channel subunit in primary cultures of neurons expressing a genetically encoded reporter of SV turnover. Knockdown of MCU resulted in ablation of activity-dependent mitochondrial calcium uptake but had no effect on the rate or extent of SV exocytosis. In contrast, the rate of SV endocytosis was increased in the absence of mitochondrial calcium uptake and slowed when MCU was overexpressed. MCU knockdown did not perturb activity-dependent increases in presynaptic free calcium, suggesting that SV endocytosis may be controlled by calcium accumulation and efflux from mitochondria in their immediate vicinity.     

Stimulation of Calcium Uptake in PC12 Cells by the Dihydropyridine Agonist BAY K 8644

Abstract: Methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate (BAY K 8644), an analog of dihydropyridine calcium channel antagonists, stimulated ⁴⁵Ca uptake into PC12 pheochromocytoma cells. Half-maximal stimulation occurred at 80 n M BAY K 8644. Enhancement of uptake was inhibited by cationic and organic calcium channel blockers, but not by tetrodotoxin, which is consistent with an effect on voltage-dependent calcium channels. Stimulation of ⁴⁵Ca uptake by BAY K 8644 occurred only at elevated concentrations of extracellular K⁺, suggesting that BAY K 8644 may interact with calcium channels in the open (activated) state.     

Pivotal Role of Mitochondrial Calcium Uptake in Neural Cell Apoptosis and Necrosis

Abstract : Perturbed cellular calcium homeostasis has been implicated in both apoptosis and necrosis, but the role of altered mitochondrial calcium handling in the cell death process is unclear. The temporal ordering of changes in cytoplasmic ([Ca²⁺]C) and intramitochondrial ([Ca²⁺]M) calcium levels in relation to mitochondrial reactive oxygen species (ROS) accumulation and membrane depolarization (MD) was examined in cultured neural cells exposed to either an apoptotic (staurosporine ; STS) or a necrotic (the toxic aldehyde 4-hydroxynonenal ; HNE) insult. STS and HNE each induced an early increase of [Ca²⁺]C followed by delayed increase of [Ca²⁺]M. Overexpression of Bcl-2 blocked the elevation of [Ca²⁺]M and the MD in cells exposed to STS but not in cells exposed to HNE. The cytoplasmic calcium chelator BAPTA-AM and the inhibitor of mitochondrial calcium uptake ruthenium red prevented both apoptosis and necrosis. STS and HNE each induced mitochondrial ROS accumulation and MD, which followed the increase of [Ca²⁺]M. Cyclosporin A prevented both apoptosis and necrosis, indicating critical roles for MD in both forms of cell death. Caspase activation occurred only in cells undergoing apoptosis and preceded increased [Ca²⁺]M. Collectively, these findings suggest that mitochondrial calcium overload is a critical event in both apoptotic and necrotic cell death.     

Calcium Accumulation by Corn Mitochondria

Corn mitochondria carry out energy-linked Ca^{2 +} accumulationwhen energized in KC1, and Ca^{2 +} release occurs when respirationceases. Stoichiometric H⁺ ejection occurs giving a H⁺: Ca^{2 +}ratio of 0.9 but insensitivity to holmium suggests lack of aCa^{2 +}-specific carrier. Non-energized corn mitochondria contain168 nmol endogenous inorganic phosphate per mg protein, about70 per cent of which is labile to Millipore filtration but stableto centrifugal filtration. Energized mitochondria, collectedby Millipore filtration, show an apparent increase in endogenousinorganic phosphate content concurrent with Ca²⁺ accumulation,both of which are inhibited by mersalyl. Accumulation of Ca²⁺also occurs in a potassium acetate medium but without involvementof endogenous Pi. Subsequent Ca²⁺ release occurs despite continuingsubstrate oxidation and is associated with a reduced abilityto synthesize adenosine triphosphate.     

Enhancement of Nitrate Uptake and Growth of Barley Seedlings by Calcium under Saline Conditions

The effect of Ca²⁺ on NO₃⁻ assimilation in young barley (Hordeum vulgare L. var CM 72) seedlings in the presence and absence of NaCl was studied. Calcium increased the activity of the NO₃⁻ transporter under saline conditions, but had little effect under nonsaline conditions. Calcium decreased the induction period for the NO₃⁻ transporter under both saline and nonsaline conditions but had little effect on its apparent K_m for NO₃⁻ both in the presence and absence of NaCl. The enhancement of NO₃⁻ transport by Ca²⁺ under saline conditions was dependent on the presence of Ca²⁺ in the uptake solution along with the salt, since Ca²⁺ had no effect when supplied before or after salinity stress. Although Mn²⁺ and Mg²⁺ enhanced NO₃⁻ uptake under saline conditions, neither was as effective as Ca²⁺. In longer studies, increasing the Ca²⁺ concentration in saline nutrient solutions resulted in increases in NO₃⁻ assimilation and seedling growth.     

Calcium Uptake by Potato Tuber Mitochondria

An attempt was made to isolate from potato tuber mitochondria the reported calcium-45 complex. Mitochondria fractionation results showed that the lipids were not involved in the calcium-45 complex formation. Using hot water as an extracting solvent it was found that two calcium-45 complexes occurred in the mitochondria fraction. One of the complexes was readily removable while the other complex was much less readily extractable with hot water. The readily removable calcinm-45 complex was identified as calcium phosphate and the less readily extractable complex seemed to be directly related to mitochondrial RNA liberation, however, it was impossible to isolate a calcium-45 ribonucleic acid complex. The extraction of the slowly removable calcium-45 complex was not a simple liberation of the complex but rather a breakdown which was irreversible and could be inhibited partially by sodium arsenate. A possible explanation of these data is that the TCA soluble fraction represents the total calcium45 uptake by the mitochondria, that is, the calcium-45 that has been accumulated plus that which is in the “calcium-carrier complex”. It is suggested that the easily extractable hot water fraction is the accumulated calicum-45 while the less readily extractable fraction is the “calcium-carrier complex”.     

Stimulation of Growth and Nutrient Uptake by VAM Fungi in Brassica Oleracea Var. Capitata

Cabbage (Brassica oleracea, var. capitata, cv. Hercules) seedlings were inoculated with vesicular-arbuscular mycorrhizal (VAM) fungi Glomus fasciculatum, G. aggregatum, and G. mosseae. Differential efficiency in mycorrhizal colonization and the specificity of fungal symbiont to stimulate the growth and nutrient uptake of the host were observed. In addition, there was an increase in phenol, protein, reducing sugar contents, and peroxidase activity in the VAM inoculated seedlings. Since these compounds are known to confer resistance against fungal pathogens, the use of VAM as a biological control agent to protect cabbage against several root diseases is suggested.     

Light Regulation of the Growth Response in Corn Root Gravitropism

Roots of Merit variety corn (Zea mays L.) require red light for orthogravitropic curvature. Experiments were undertaken to identify the step in the pathway from gravity perception to asymmetric growth on which light may act. Red light was effective in inducing gravitropism whether it was supplied concomitant with or as long as 30 minutes after the gravity stimulus (GS). The presentation time was the same whether the GS was supplied in red light or in darkness. Red light given before the GS slightly enhanced the rate of curvature but had little effect on the lag time or on the final curvature. This enhancement was expanded by a delay between the red light pulse and the GS. These results indicate that gravity perception and at least the initial transduction steps proceed in the dark. Light may regulate the final growth (motor) phase of gravitropism. The time required for full expression of the light enhancement of curvature is consistent with its involvement in some light-stimulated biosynthetic event.     

Calcium Uptake by a Psychrophilic Achromobacter Strain

The permeability of a psychrophilic Achromobacter strain to calcium ions was examined with the radioisotope ⁴⁵Ca²⁺. The amount of radioactivity that remained associated with the cells after exposure to 5 to 200 mM solutions of radioactive calcium was determined. The concentration of ⁴⁵Ca²⁺ of the cells compared to that of the surrounding medium gave a ratio larger than one, and this ratio increased with decreasing ambient calcium concentration. The same results showed that the higher the external calcium concentration was, the more calcium remained with the cells. The radioactivity of the cells had the following characteristics: 1. It was rapidly lost when the cells were washed with ⁴⁰Ca-solution. 2. Most of it was retained after water washings. 3. It was not affected by the presence of 2,4-dinitrophenol. 4. It was only slightly affected by temperature. 5. It increased when the cells were treated with toluene or heat and reached a maximum of 2–3 times the value of untreated cells. The same treatment brought about a smaller increase of the uptake of ²²Na⁺. 6. Approximately 60% of the radioactivity of whole cells was associated with isolated cell envelopes.     

Stimulation of Shoot Regeneration on Linum Hypocotyl Segments by Thidiazuron and Its Response to Light and Calcium

The basal segment from hypocotyl of Linum usitatissimum L. seedling readily regenerates, to produce a large number of shoots, in a short period of 5 [ndash ] 7 d. This response was stimulated by a low concentration (0.1 [micro ]M) of thidiazuron (TDZ). TDZ was also effective in inducing regeneration in dark. A drastic reduction in regeneration response on hormone-free as well as TDZ-supplemented medium was found after inclusion of an inhibitor of calcium-uptake, lanthanum (La³⁺). An essentiality of calcium in the regeneration was also evidenced by an increased response with increasing concentration of calcium. At Ca²⁺ concentration insufficient for regeneration, inclusion of TDZ resulted in shoot formation.     

Inhibition of Cottonseed Choline- and Ethanolaminephosphotransferases by Calcium during Postgerminative Growth

Activities of choline- and ethanolaminephosphotransferase (CPT and EPT) were reproducibly high in microsomes from imbibed seeds of cotton (Gossypium hirsutum, L.). Initial studies showed that both activities dramatically declined during postgerminative growth when demand for phosphatidylcholine (PC) and phosphatidylethanolamine (PE) synthesis was high. Addition of CaCl₂ (0.1 millimolar) or aliquots of supernatant fractions (150,000g, 60 minutes) from cotyledons of 48-hour-old seedlings to imbibed-seed microsomes reduced the CPT and EPT activities to levels approximating those found in 48-hour microsomes. Inhibition by supernatants was completely reversed by adding EGTA (1.0 millimolar), but not by boiling the supernatants. EGTA (1.0 or 5.0 millimolar) relieved inhibition in cellular fractions whether it was added to the homogenization media or the assay reaction mixtures. A time course of CPT and EPT activities in cellular fractions prepared with 1.0 millimolar EGTA showed that activities were well developed in imbibed seeds, doubled coincidentally to a peak at 36 hours, then declined during the next 12 hours to levels approximating those in imbibed seeds. Greater than 90% of the CPT and EPT activities were pelletable (150,000g, 60 minutes) at all ages examined. Calcium apparently was artificially released upon homogenization, to a progressively greater extent in older cotyledons, and severely inhibited CPT and EPT activities. This is the only time course of CPT and EPT activities reported for cotyledons of any oilseed; it is substantially different from that in oil-storing endosperm.     

Cyclosporin a Increases Mitochondrial Calcium Uptake Capacity in Cortical Astrocytes but not Cerebellar Granule Neurons

Isolated brain mitochondria are a heterogeneous mixture from different cell types and these subsets may have differing sensitivities to Ca²⁺-induced membrane permeability transition (MPT) and to inhibition of the MPT by cyclosporin A (CsA). This study tested the hypothesis that mitochondria within primary cultures of astrocytes and neurons exhibit different energy-dependent Ca²⁺ uptake capacities and different degrees to which CsA increases their uptake capacity. Astrocytes and neurons were suspended in a cytosol-like medium containing respiratory substrates, ATP, and Mg²⁺ in the presence of digitonin to selectively permeabilize the plasma membrane. Uptake of added Ca²⁺ by mitochondria within the cells was measured by Calcium Green 5N fluorescent monitoring of the medium [Ca²⁺]. Permeabilized astrocytes had a fourfold higher Ca²⁺ uptake capacity, relative to neurons and a twofold higher content based on relative contents of mitochondria assessed by measurements of mitochondrial DNA and cytochrome oxidase subunit 1 protein. In astrocytes the Ca²⁺ uptake capacity was increased twofold by preincubation with 2–5 μM CsA, while in neurons CsA had no effect. Similar results were obtained using measurements of the effects of added Ca²⁺ on mitochondrial membrane potential. FK506, a drug similar to CsA but without MPT inhibitory activity, had no effect on either cell type. These results are consistent with the presence of a calcium-induced MPT in astrocytes, even in the presence of ATP, and indicate that the MPT in cerebellar granule neurons is resistant to CsA inhibition. Some of the protective effects of CsA in vivo may therefore be mediated by preservation of mitochondrial functional integrity within astrocytes.     

Stimulation of Root Elongation and Curvature by Calcium

Ca²⁺ has been proposed to mediate inhibition of root elongation. However, exogenous Ca²⁺ at 10 or 20 millimolar, applied directly to the root cap, significantly stimulated root elongation in pea (Pisum sativum L.) and corn (Zea mays L.) seedlings. Furthermore, Ca²⁺ at 1 to 20 millimolar, applied unilaterally to the caps of Alaska pea roots, caused root curvature away from the Ca²⁺ source, which was caused by an acceleration of elongation growth on the convex side (Ca²⁺ side) of the roots. Roots of an agravitropic pea mutant, ageotropum, responded to a greater extent. Roots of Merit and Silver Queen corn also responded to Ca²⁺ in similar ways but required a higher Ca²⁺ concentration than that of pea roots. Roots of all other cultivars tested (additional four cultivars of pea and one of corn) curved away from the unilateral Ca²⁺ source as well. The Ca²⁺-stimulated curvature was substantially enhanced by light. A Ca²⁺ ionophore, A23187, at 20 micromolar or abscisic acid at 0.1 to 100 micromolar partially substituted for the light effect and enhanced the Ca²⁺-stimulated curvature in the dark. Unilateral application of Ca²⁺ to the elongation zone of intact roots or to the cut end of detipped roots caused either no curvature or very slight curvature toward the Ca²⁺. Thus, Ca²⁺ action on root elongation differs depending on its site of application. The stimulatory action of Ca²⁺ may involve an elevation of cytoplasmic Ca²⁺ in root cap cells and may participate in root tropisms.     

Effect of pH on Orthophosphate Uptake by Corn Roots

Orthophosphate (Pi) influx in washed corn roots was studied with experimental conditions allowing a distinction of pH effects on Pi ionization in the medium and on the transport system itself. There appeared to be no relationship between the pH dependencies of membrane potential, H⁺ secretion, and ³²Pi influx. The Pi uptake versus pH curves were compared to the calculated ones describing the concentrations of the different ionized Pi forms in the medium and in the cell walls; the latter were obtained using the theoretical model described by Sentenac and Grignon (1981) Plant Physiol 68: 415-419). The conclusion was that the transported form is H₂PO₄⁻ and the concentration sensed by the transport system is the local one. The ionic compositions of experimental media were manipulated to ensure constant pH and various H₂PO₄⁻ concentrations, or constant H₂PO₄⁻ concentration and various pH values in the walls. The kinetic analysis of the results in the micromolar range showed that the transport system has an intrinsic sensitivity to pH, and is switched from a low activity state at pH > 6 to a high activity one at pH < 4 (pH in the walls). This change could be triggered by the protonation of a group with pK 5.5.     

Whole-Brain Calcium Imaging during Physiological Vestibular Stimulation in Larval Zebrafish

1. Download high-res image (177KB)
2. Download full-size image

     

Suppression of Calcium Transport to Shoots by Root Restriction in Tomato Plants

Restriction of tomato roots by growth in small containers stronglysuppressed transport of ⁴⁵Ca²⁺ ions to new leaves and apices.Water transport, expressed on a leaf area basis, was marginallyreduced by root restriction, an indication that calcium transportwas more severely limited than water transport. (Received October 11, 1996; Accepted January 27, 1997)     

Arbuscular Mycorrhizae Effects on Heavy Metal Uptake by Corn

Maximizing uptake of soil-borne metals into plants is important for successful phytoremediation. Arbuscular mycorrhizae (AM) have been shown previously by our group to sequester metals in the roots of plants and prevent translocation to the shoot. If AM colonization of roots can be reduced, it may be possible to increase metal uptake into plants, thus increasing the efficiency of phytoremediation. The fungicide Benomy® was applied to a Pb-contaminated soil and seeded to corn (Zea mays). Because soil pH affects metal solubility, two pHs were also examined. Colonization of roots by AM was significantly decreased by application of Benomyl to soil, but only at the higher soil pH. Benomyl increased the concentration of several elements, including Pb, in shoots. However, the total Pb content in the shoot decreased due to the reduced shoot weight associated with Benomyl application.

     

Growth Substance Interactions during Uptake by Mesocotyl Segments of Zea mays L.

The effects of various growth substances on the ‘metabolic’uptake of indol-3yl-acetic acid (IAA) by Zea mesocotyl segmentswas investigated using methods of fluorescence spectroscopyand radioactivity assay. 2, 4-Dichlorophenoxyacetic acid (2,4-D) and -(I-naphthylmethylthio)propionic acid (NMSP) exertedno discernible effects on IAA uptake, whereas N-I-naphthylphthalmicacid (NPA) stimulated uptake to some degree. Low concentrationsof 2,3,5-tri-iodobenzoic acid (TIBA) promoted the uptake oflow IAA concentrations, while higher concentrations were decidedlyinhibitory. 2,4-Dinitrophenol (DNP), ioxynil, and bromoxynilalso induced marked inhibition presumably by preventing oxidativephosphorylation. The uptake interactions between these compoundswere examined in relation to concentration and time. In no casewas there evidence of competitive interaction. The inhibitoryeffects of TIBA on IAA uptake were considerably greater thanthose of DNP. SH-enzyme protectors such as BAL and cysteinedid not relieve these inhibitions. The absorption of TIBA-¹³¹Iwas completely unaffected by any concentration of IAA tested.Chromatographic and radio-autographic analysis revealed no detectableproducts of IAA-I-¹⁴C metabolism or degradation in maize mesocotyltissue during the 6-h experimental period and this was not alteredby TIBA treatment. Respiratory decarboxylation of IAA-I-¹⁴Cwas found to be negligible and unaffected by TIBA.