Flufenamic acid as an inducer of mitochondrial permeability transition

To assess the mechanism by which mitochondrial permeability transition (MPT) is induced by the nonpolar carboxylic acids, we investigated the effects of flufenamic acid (3-trifluoromethyl diphenylamine-2-carboxylic acid, FA) on mitochondrial respiration, electrical transmembrane potential difference (), osmotic swelling, Ca²⁺ efflux, NAD(P)H oxidation and reactive oxygen species (ROS) generation. Succinate-energized isolated rat liver mitochondria incubated in the absence or presence of 10 M Ca²⁺, 5 M ruthenium red (RR) or 1 M cyclosporin A (CsA) were used. The dose response-curves for both respiration release and dissipation were nearly linear, presenting an IC₅₀ of approximately 10 M and reaching saturation within 25-50 M, indicating that FA causes mitochondrial uncoupling by a protonophoric mechanism. Within this same concentration range FA showed the ability to induce MPT in energized mitochondria incubated with 10 M Ca²⁺, followed by dissipation and Ca²⁺ efflux, and even in deenergized mitochondria incubated with 0.5 mM Ca²⁺. ADP, Mg²⁺, trifluoperazine (TFP) and N-ethylmaleimide (NEM) reduced the extent of FA-promoted swelling in energized mitochondria by approximately one half, whereas dithiothreitol (DTT) slightly enhanced it. NAD(P)H oxidation and ROS generation (H₂O₂ production) by mitochondria were markedly stimulated by FA; these responses were partly prevented by CsA, suggesting that they may be implicated as both a cause and effect of FA-induced MPT. FA incubated with mitochondria under swelling assay conditions caused a decrease of approximately 40% in the content of protein thiol groups reacting with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB). The present results are consistent with a ROS-intermediated sensitization of MPT by a direct or indirect FA interaction with inner mitochondrial membrane at a site which is in equilibrium with the NAD(P)H pool, namely thiol groups of integral membrane proteins.     

Na+/K+-ATPase inhibition during cardiac myocyte swelling: Involvement of intracellular pH and Ca2+

Previous studies in chick embryo cardiac myocytes have shown that the inhibition of Na⁺/K⁺-ATPase with ouabain induces cell shrinkage in an isosmotic environment (290 mOsm). The same inhibition produces an enhanced RVD (regulatory volume decrease) in hyposmotic conditions (100 mOsm). It is also known that submitting chick embryo cardiomyocytes to a hyperosmotic solution induces shrinkage and a concurrent intracellular alkalization. The objective of this study was to evaluate the involvement of intracellular pH (pH_i), intracellular Ca²⁺ ([Ca²⁺]_i) and Na⁺/K⁺-ATPase inhibition during hyposmotic swelling. Changes in intracellular pH and Ca²⁺ were monitored using BCECF and fura-2, respectively. The addition of ouabain (100 M) under both isosmotic and hyposmotic stimuli resulted in a large increase in [Ca²⁺]_i (200%). A decrease in pH_i (from 7.3 ± 0.09 to 6.4 ± 0.08, n = 6; p < 0.05) was only observed when ouabain was applied during hyposmotic swelling. This acidification was prevented by the removal of extracellular Ca²⁺. Inhibition of Na⁺/H²⁺ exchange with amiloride (1 mM) had no effect on the ouabain-induced acidification. Preventing the mitochondrial accumulation of Ca²⁺ using CCCP (10 M) resulted in a blockade of the progressive acidification normally induced by ouabain. The inhibition of mitochondrial membrane K⁺/H⁺ exchange with DCCD (1 mM) also completely prevented the acidification. Our results suggest that intracellular acidification upon cell swelling is mediated by an initial Ca²⁺ influx via Na⁺/Ca²⁺ exchange, which under hyposmotic conditions activates the K⁺ and Ca²⁺ mitochondrial exchange systems (K⁺/H⁺ and Ca²⁺/H⁺).Deceased     

Kinetic Analysis of the Calcium- and Cadmium-Induced Development of Nonspecific Permeability of the Mitochondrial Inner Membrane

We studied the Са²⁺- and Cd²⁺-induced development of the nonspecific permeability of the mitochondrial inner membrane in preparations obtained from rat liver tissue, which is accompanied by swelling of these organelles and intensification of light dispersion of their suspension. Addition of 5 to 100 μM Са²⁺ or 1 to 50 μM Сd²⁺ to the medium caused swelling of the mitochondria. With increase in concentrations of Са²⁺ and Cd²⁺, the latency of the effect decreased, and the rate of swelling of these organelles increased. Upon isolated action of Са²⁺, the intensity of the process (amplitude of changes) did not depend significantly on the concentration of the above ions, while upon isolated action of Cd²⁺, it was the maximum at the concentration of 1 mM and noticeably decreased with increase in the concentration. The dependence of the rate of Са²⁺- and Cd²⁺-induced swelling of the mitochondria on the concentration of these ions was described by power and sigmoid functions, respectively. The calculated maximum rate and the constant of 50% saturation of these processes were equal to 0.609 and 1.084 extinction units/min⋅mg protein and 19.85 and 7.28 μM for Са²⁺- and Cd²⁺-induced swelling of the mitochondria, respectively. Cyclosporine A (10 μM) suppressed completely the Са²⁺-induced swelling of the mitochondria and decreased only partly the Cd²⁺-induced swelling. Dithiothreitol (1 mM) inhibited completely the latter effect but did not influence significantly the Са²⁺-stimulated process. Therefore, the distinctions between the kinetics of Са²⁺- and Cd²⁺-induced swelling of the mitochondria, as well as the different sensitivity of these processes to cyclosporine A and dithiothreitol, prove that the mechanisms underlying interactions between the cations of the above metals and the inner mitochondrial membrane in the course of the development of nonspecific permeability of these organelles are dissimilar. *Deceased     

Effects of cadmium accumulation on growth and respiration of a cadmium-sensitive strain of Bacillus subtilis and a selected cadmium resistant mutant

The effects of cadmium on the growth and respiration of two strains of Bacillus subtilis are compared to the accumulation of Cd by viable and cyanide-killed cells, protoplasts and cell fractions of the strains. Growth and respiration of strain 1A1 were significantly inhibited at 10g Cd²⁺/ml while the growth and respiration of strain 1A1R, a selected mutant of 1A1, were only slightly affected. Similarly, 1A1R protoplasts were more resistant to Cd than were 1A1 protoplasts. The differential resistance of the strains correlates with the accumulation of Cd by the two strains, with 1A1 accumulating approximately 10 times the level of Cd after a 4 h exposure to 1 g Cd²⁺/ml. The distributions of Cd throughout the cells, however, were similar between strains. Based on the accumulation of Cd by cyanide-killed protoplasts, uptake of Cd by 1A1 appears to be an active process, while for 1A1R, Cd accumulation is independent of protoplast viability.Non-standard abbreviations SMM Subtilis Minimal Medium - AAS Atomic Absorption Spectrophotometry - TSA Trypticase Soy Agar - PCA Plate Count Agar - INT 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl-2H tetrazolium chloride - dd H₂O double distilled demineralized water - OD Optical Density     

Processes Maintaining Calcium Homeostasis in Acinar Cells of the Rat Submandibular Salivary Gland

Using a Ca²⁺-sensitive fluorescent indicator, fura-2/AM, we recorded calcium transients in secretory cells of isolated acini of the rat submandibular salivary gland; these transients were induced by hyperpotassium-induced depolarization (after an increase in [K⁺] _e up to 50 mM) of the plasma membrane of the above cells. Calcium transients were significantly suppressed by 50 M nifedipine. Addition of 10 M carbonyl cyanide m-chlorophenylhydrazone to the normal extracellular solution was accompanied by a rise in [Ca²⁺] _i , whereas when hyperpotassium solution is used the effect was less expressed. Blockers of CA²⁺-ATPase in the cellular membrane and in the endoplasmic reticulum, eosin Y (5 M) and cyclopiazonic acid (CPA, 5 M), respectively, evoked a significant increase in [Ca²⁺] _i and a decrease in the K⁺-depolarization-induced calcium transient. Extracellular application of caffeine (2, 10, or 30 mM) was accompanied by a concentration-dependent rise in [Ca²⁺] _i . Therefore, potassium depolarization of the plasma membrane of acinar cells of the rat submandibular salivary gland activates both the voltage-dependent Ca²⁺ influx and Ca²⁺-induced Ca²⁺ release from the endoplasmic reticulum; the initial level of [Ca²⁺] _i was restored at the joint involvement of Ca²⁺-ATPases in the plasma membrane and the membranes of the endoplasmic reticulum and mitochondria.     

Alterations in calcium homeostasis on lead exposure in rat synaptosomes

The effects of lead on Ca²⁺ homeostasis in nerve terminals was studied. Incubation with leadin vitro stimulated the activity of calmodulin and the maximum effect was observed at 30 M lead, higher concentrations had an inhibitory effect.In vivo exposure to lead increased the activity of calmodulin by 45%. Lead had an inhibitory effect on Ca²⁺ ATPase activity in both calmodulin-rich and calmodulin-depleted synaptic plasma membranes, the IC₅₀ values for inhibition being 13.34 and 16.69 M respectively. Exogenous addition of calmodulin (5 g) and glutathione (1 mM) to calmodulin rich synaptic plasma membranes reversed the inhibition by IC₅₀ concentration of lead.In vivo exposure of lead also significantly reduced the Ca²⁺ ATPase activity, resulting in an increase in intrasynaptosomal calcium. Concomitant with the increase in intrasynaptosomal calcium, lipid peroxidation values also increased significantly in lead-treated animals. In addition lead also had an inhibitory effect on depolarization induced Ca²⁺ uptake and the inhibition was found to be a competitive one. The results sugest that lead exerts its toxic effects by modifications of the intracellular calcium messenger system which would have serious consequences on neuronal functioning.     

Regulation by Magnesium of Potato Tuber Mitochondrial Respiratory Activities

Dehydrogenase activities of potato tuber mitochondria and corresponding phosphorylation rates were measured for the dependence on external and mitochondrial matrix Mg²⁺. Magnesium stimulated state 3 and state 4 respiration, with significantly different concentrations of matrix Mg²⁺ required for optimal activities of the several substrates. Maximal stimulation of respiration with all substrates was obtained at 2-mM external Mg²⁺. However, respiration of malate, citrate, and -ketoglutarate requires at least 4-mM Mg²⁺ inside mitochondria for maximization of dehydrogenase activities. The phosphorylation system, requires a low level of internal Mg²⁺ (0.25 mM) to reach high activity, as judged by succinate-dependent respiration. However, mitochondria respiring on citrate or -ketoglutarate only sustain high levels of phosphorylation with at least 4-mM matrix Mg²⁺. Respiration of succinate is active without external and matrix Mg²⁺, although stimulated by the cation. Respiration of -ketoglutarate was strictly dependent on external Mg²⁺ required for substrate transport into mitochondria, and internal Mg²⁺ is required for dehydrogenase activity. Respiration of citrate and malate also depend on internal Mg²⁺ but, unlike -ketoglutarate, some activity still remains without external Mg²⁺. All the substrates revealed insensitive to external and internal mitochondrial Ca²⁺, except the exogenous NADH dehydrogenase, which requires either external Ca²⁺ or Mg²⁺ for detectable activity. Calcium is more efficient than Mg²⁺, both having cumulative stimulation. Unlike Ca²⁺, Mn²⁺ could substitute for Mg²⁺, before and after addition of A23, showing its ability to regulate phosphorylation and succinate dehydrogenase activities, with almost the same efficiency as Mg²⁺.     

On the involvement of cyclic AMP and extracellular Ca2+ in the regulation of hormone release from rat pituitary tumour (GH3) cells in culture

Thyroliberin (TRH), dibutyryl cyclic AMP (db-cAMP), and 3-isobutyl-l-methylxanthine (MIX) had a stimulatory effect on prolactin (PRL) and growth hormone (GH) release from GH 3 cells. Half-maximal and maximal effects were observed for TRH at 2.5 nM and 10 nM; for db-cAMP at 0.6 mM and 5 mM, respectively. MIX (0.1 mM–1 mM) induced a dose-dependent accumulation of cellular cyclic AMP, while the hormone release was already maximally stimulated at 0.1 mM MIX. The maximal effects on hormone release of TRH and db-cAMP, but not of TRH and MIX, were additive.The Ca²⁺ channel blockers Co²⁺ (5 mM) and verapamil (100 M) and the Ca²⁺ chelator EGTA (4 mM) abolished the stimulatory effect of TRH (1 M) on hormone release. Co²⁺ and verapamil, but not EGTA, inhibited the stimulatory effect of db-cAMP (5 mM) on hormone release. The inhibitory effects of Co²⁺ and verapamil on GH release were counteracted by the combination of TRH and db-cAMP. For PRL release Co²⁺, but not verapamil, was able to inhibit the combined action of TRH and db-cAMP. Co²⁺, verapamil, and EGTA eliminated the stimulatory effect of MIX (1 mM) on PRL release while only Co²⁺ and EGTA affected the GH release. Hormone release in the presence of MIX plus verapamil or EGTA, but not Co²⁺, was increased by TRH.The calmodulin antagonist trifluoperazine (TFP) at 30 M inhibited basal hormone release and hormone release stimulated by TRH (1 M), db-cAMP (5 mM), and MIX (1 mM). The Ca²⁺ ionophore A23187 (5 M) had a stimulatory effect on basal hormone release which was abolished by 30 M TFP.     

Effects of heavy metals on pollen tube growth and ultrastructure

Summary The influence of different concentrations of the heavy metals cadmium (Cd²⁺), cobalt (Co²⁺), copper (Cu²⁺), iron (Fe²⁺ and Fe³⁺), mercury (Hg²⁺), manganese (Mn²⁺), and zinc (Zn²⁺), plus aluminium (Al³⁺) (a toxic metal in polluted areas), on pollen germination and tube growth ofLilium longiflorum was investigated using light microscopy. Effects could be observed with 3 M and 100 M of heavy metal, added as chloride salts to the medium. Cd²⁺, Cu²⁺, and Hg²⁺, showed the greatest toxicity, whereas germination and growth rate was less affected by Mn²⁺. Affected tubes showed swelling of the tip region. Tubes treated with Cd²⁺, Co²⁺, Fe²⁺, Fe³⁺, Hg²⁺, and Mn²⁺ were also prepared for ultrastructural studies. In all cases, the main effect was abnormal cell wall organization, mostly at the tip, where round, fibrillar aggregates, the shape and size of secretory Golgi vesicles were formed. They built up a loose network which could be up to 10 m thick compared to untreated tubes where the cell wall was composed of thin layers of long fibrils and about 100 nm thick. Cd²⁺ was the only metal which produced effects at the intracellular level: organelle distribution within the tip region appeared disorganized. A general mechanism of heavy metal action on pollen tube growth is discussed.     

In Saccharomyces cerevisiae,cations control the fate of the energy derived from oxidative metabolism through the opening and closing of the yeast mitochondrial unselective channel

The yeast mitochondrial unspecific channel (YMUC) sensitivity to inorganic (Ca²⁺ or Mg²⁺) or organic (hexyl or octyl-guanidine) cations was measured. The rate of oxygen consumption in State 3 and State 4, the transmembrane potential (), mitochondrial swelling, and the polyethylene-glycol mediated recontraction were used to follow opening of the YMUC. Addition of 0.4 mM PO₄ did not close the YMUC, although it did enhance the sensitivity to Ca²⁺ (I₅₀ decreased from 50 to 0.3 mM) and Mg²⁺ (I₅₀ decreased from 5 to 0.83 mM Mg²⁺). The Ca²⁺ concentration needed to close the YMUC was higher than the concentrations usually observed in the cell. Nonetheless, Mg²⁺, Ca²⁺, and PO₄ exhibited additive effects. These cations did not inhibit contraction of preswollen mitochondria, suggesting that the YMUC/cation interaction was labile. Octyl-guanidine (OG-I₅₀ 7.5 M) was the only cation which inhibited mitochondrial recontraction, probably as a result of membrane binding stabilization through its hydrophobic tail. The PO₄-dependent, Ca²⁺/Mg²⁺-mediated closure of the YMUC may be a means to control the proportion of oxidative energy producing ATP or being lost as heat.     

Involvement of Ca2+-stimulated adenosine 5′-triphosphatase in the Ca2+ releasing mechanism of rat liver nuclei

The regulatory role of Ca²⁺-stimulated adenosine 5-triphosphatase (Ca²⁺-ATPase) in Ca²⁺ transport system of rat liver nuclei was investigated. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺–Mg²⁺)-ATPase activity. The release of Ca²⁺ from the Ca²⁺-loaded nuclei was evoked progressively after Ca²⁺ uptake with 1.0 mM ATP addition, while it was only slightly in the case of 2.0 mM ATP addition, indicating that the consumption of ATP causes a leak of Ca²⁺ from the Ca²⁺-loaded nuclei. The presence of N-ethylmaleimide (NEM; 0.1 mM) caused an inhibition of nuclear Ca²⁺ uptake and induced a promotion of Ca²⁺ release from the Ca²⁺-loaded nuclei. NEM (0.1 and 0.2 mM) markedly inhibited nuclear Ca²⁺-ATPase activity. This inhibition was completely blocked by the presence of dithiothreitol (DTT; 0.1 and 0.5 mM). Also, DTT inhibited the effect of NEM (0.1 mM) on nuclear Ca²⁺ uptake and release. Meanwhile, verapamil and diltiazem (10 M), a blocker of Ca²⁺ channels, did not prevent the NAD⁺ (1.0 and 2.0 mM), zinc sulfate (1.0 and 2.5 M) and arachidonic acid (10 M)-induced increase in nuclear Ca²⁺ release, suggesting that Ca²⁺ channels do not involve on Ca²⁺ release from the nuclei. These results indicates that an inhibition of nuclear Ca²⁺-ATPase activity causes the decrease in nuclear Ca²⁺ uptake and the release of Ca²⁺ from the Ca²⁺-loaded nuclei. The present finding suggests that Ca²⁺-ATPase plays a critical role in the regulatory mechanism of Ca²⁺ uptake and release in rat liver nuclei.     

Ecophysiological characterization of carnivorous plant roots: Oxygen fluxes,respiration, and water exudation

Various ecophysiological investigations on carnivorous plants in wet soils are presented. Radial oxygen loss from roots of Droseraceae to an anoxic medium was relatively low 0.02 – 0.07 mol(O₂) m^{– 2} s^–1 in the apical zone, while values of about one order of magnitude greater were found in both Sarracenia rubra roots and Genlisea violacea traps. Aerobic respiration rates were in the range of 1.6 – 5.6 mol kg^–1 (f.m.) s^–1 for apical root segments of seven carnivorous plant species and 0.4 – 1.1 mol kg^–1 (f.m.) s^–1 for Genlisea traps. The rate of anaerobic fermentation in roots of two Drosera species was only 5 – 14 % of the aerobic respiration. Neither 0.2 mM NaN₃ nor 0.5 mM KCN influenced respiration rate of roots and traps. In all species, the proportion of cyanide-resistant respiration was high and amounted to 65 – 89 % of the total value. Mean rates of water exudation from excised roots of 12 species ranged between 0.4 – 336 mm 3 kg^–1 (f.m.) s^–1 with the highest values being found in the Droseraceae. Exudation from roots was insensitive to respiration inhibitors. No significant difference was found between exudation rates from roots growing in situ in anoxic soil and those kept in an aerated aquatic medium. Carnivorous plant roots appear to be physiologically very active and well adapted to endure permanent soil anoxia.     

Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line

Summary We have investigated muscarinic receptor-operated Ca²⁺ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca²⁺ release and extracellular Ca²⁺ entry. The carbachol (Cch) dose response of intracellular Ca²⁺ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K _0.510 or 30 m, depending on the method for [Ca²⁺] _i determination). However, similar data for Ca²⁺ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca²⁺ release and a second higher affinity site withK _0.52.5 m. In addition, the Ca²⁺ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K⁺ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca²⁺ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca²⁺] _i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca²⁺ channel blocker La³⁺ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca²⁺ in these cells by increasing Ca²⁺ entry. Organic Ca²⁺ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca²⁺ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K⁺ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP₃) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP₃ formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca²⁺ entry in HSG-PA cells. One is associated with IP₃ formation and intracellular Ca²⁺ release and is independent of membrane potential; the other is less dependent on IP₃ formation and intracellular Ca²⁺ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.     

Enhancement of plasma membrane (Ca2+-Mg2+)-ATPase activity in regenerating rat liver: Involvement of endogenous activating protein regucalcin

The alteration of (Ca²⁺-Mg²⁺)-ATPase activity in the plasma membranes of regenerating rat liver after a partial hepatectomy was investigated. Liver was surgically removed about two thirds of that of sham-operated rats. The reduced liver weight by partial hepatectomy was restored about 50% at 24 h after the surgery, and it was completely restored at 72 h. Regenerating liver significantly increased calcium content and plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity between 12–48 h after hepatectomy. Those increases were maximum at 24 h after the surgery. The regenerating liver-induced increase in hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was completely abolished by the presence of anti-regucalcin IgG (1.0–4.0 g/ml). The regenerating liver-induced increase in hepatic plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity was clearly inhibited by N-ethylmaleimide (2.5 and 5.0 mM) addition into the enzyme reaction mixture. This NEM effect was also seen for the activatory effect with regucalcin (0.25 M) addition on the enzyme activity in the plasma membranes from normal rat liver. The endogenous regucalcin may play a cell physiological role in the activation of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase to maintain the intracellular calcium level in regenerating rat liver.     

Haloperidol reduces K+-evoked Ca2+-dependentd-[3H]aspartate release from rat hippocampal slices

Rat hippocampal slices preloaded withd-[³H]aspartate, a non metabolizable analogue ofl-glutamate, were superfused with artifical CSF. Depolarization was induced by 53.5 mM K⁺, in the presence of Ca²⁺ (1.3 mM) or Mg²⁺ (5 mM) to determine the Ca²⁺ dependent release. Haloperidol added in the superfusion medium at 100 M reduced by about 60% the Ca²⁺ dependent release ofd-[³H]aspartate. This drug at 20 M or 100 M inhibited the non-activated glutamate dehydrogenase (GDH) but had no effect on GDH activated by ADP (2 mM) or leucine (5 mM). In addition no effect was observed on phosphate activated glutaminase (PAG) in the presence either of 20 mM or 5 mM phosphate. These results indicate that the effect of haloperidol is exerted on presynaptic mechanisms regulating neurotransmitter release.     

Iron promotes cadmium binding to citrate

Ironcadmium interactions are important in cadmium toxicity. Dietary iron supplements may decrease cadmium retention after oral cadmium exposure but the underlying mechanism is not known. Using a CdS/AgS ion selective electrode to measure [Cd²⁺] in physiological saline solution at pH 7.4, we show that Fe²⁺ promotes Cd²⁺ binding to citrate thereby decreasing the availability of free Cd²⁺. This suggests the formation of high molecular weight Cd²⁺Fe²⁺citrate complexes. We confirm this suggestion by showing that ¹⁰⁹Cd²⁺ is retained by 1 kDa cut off filters when present with total 50 M Fe²⁺ plus 1 mM citrate but not when present with citrate alone. The formation of high molecular weight complexes may prevent Cd²⁺ absorption. As citrate is part of the diet, we suggest that these ironcadmium interactions may contribute to the protective effect of iron against cadmium toxicity.     

Effects of ADP upon the ATP-sensitive K+ channel in rat ventricular myocytes

Summary The effects of ADP upon the gating of ATP-sensitive K⁺ channels from rat ventricular myocytes have been investigated by patch-clamp single-channel current recording experiments. ADP was applied to the internal surface of excised insideout membrane patches and depending upon the experimental protocol and the concentration it was found that ADP could either inhibit or stimulate openings of ATP-sensitive K⁺ channels. In the absence of inactivation, ATP-sensitive K⁺ channels were inhibited by ADP in a dose-dependent manner. Partially inactivated channels, on the other hand, were stimulated by low (10 to 250 M) and inhibited by high (>250 M) concentrations of ADP. ATP-sensitive K⁺ channels which were being inhibited by ATP (<1 mM) could be opened by the simultaneous application of ADP (50 M to 1 mM). ADP had no effect upon channels inhibited by mM concentrations of ATP. The situation was further complicated when it was found that inhibition evoked by ADP was strongly attenuated by the presence of Mg²⁺ ions whilst channel stimulation, whether of partially inactivated channels or channels inhibited by ATP, required the presence of Mg²⁺ ions. The analog of ADP, ADPS, always evoked inhibition of ATP-sensitive K⁺ channels which was not affected by the presence or absence of Mg²⁺ ions.     

Effect of nuclear Ca2+ uptake inhibitors on Ca2+-activated DNA fragmentation in rat liver nuclei

The effect of nuclear Ca²⁺ uptake inhibitors on the Ca²⁺-activated DNA fragmentation in rat liver nuclei was investigated. The addition of Ca²⁺ (40 M) into the reaction mixture containing liver nuclei in the presence of 2.0 mM ATP caused a remarkable increase in nuclear DNA fragmentation. This Ca²⁺-activated DNA fragmentation was not seen in the absence of ATP, because nuclear Ca²⁺ uptake is not initiated without ATP addition. Moreover, the presence of various reagents (10 M arachidonic acid, 2.0 mM NAD⁺, 10 M zinc sulfate and 0.2 mM N-ethylmaleimide), which could inhibit Ca²⁺-ATPase activity and Ca²⁺ uptake in the nuclei, produced a significant inhibition of the Ca²⁺-activated DNA fragmentation in the nuclei. The results show that the Ca²⁺-activated DNA fragmentation is involved in the uptake of Ca²⁺ by the nuclei, suggesting a role of Ca²⁺ transport system in the regulation of liver nuclear functions.     

Regulatory effect of regucalcin on (Ca2+−Mg2+)-ATPase in rat liver plasma membranes: comparison with the activation by Mn2+ and Co2+

The effect of various metals and regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes of rat liver was investigated. Of various metals (Zn²⁺, Cu²⁺, Ni²⁺, Mn²⁺, Co²⁺ and Al³⁺; 100 M as a final concentration), Mn²⁺ and Co²⁺ increased markedly (Ca²⁺–Mg²⁺)-ATPase activity, while other metals had no effect. When Ca²⁺ was not added into enzyme reaction mixture, Mn²⁺ and Co²⁺ (25–100 M) did not significantly increase the enzyme activity, indicating that heavy metals act on Ca²⁺-stimulated phosphorylation of the enzyme. Meanwhile, regucalcin (0.25–1.0 M) caused a remarkable elevation of (Ca²⁺–Mg²⁺)-ATPase activity. This increase was not inhibited by the presence of 100 M vanadate, although the effects of Mn²⁺ and Co²⁺ (100 M) were inhibited by vanadate. Also, the inhibition of the Mn²⁺ and Co²⁺ effects by vanadate was not seen in the presence of regucalcin. Moreover, regucalcin (0.5 M) increased significantly the enzyme activity in the absence of Ca²⁺. This effect of regulcalcin was not altered by increasing concentrations of Ca²⁺ added, indicating that the regucalcin effect does not depend on Ca²⁺. The present results suggest that regucalcin activates directly (Ca²⁺–Mg²⁺)-ATPase in liver plasma membranes, and that the activation is not involved in the Ca²⁺-dependent phosphorylation of the enzyme.     

2,3-Dimercaptopropanol Inhibits Ca2+ Transport in Microsomes from Brain But Not from Fast-Skeletal Muscle

Ca²⁺ is involved in the regulation of a variety of physiological processes, but a persistent increase in free cytosolic Ca²⁺ concentrations may contribute to cell injury. Dimercaprol (BAL) is a compound used in the treatment of mercury intoxication, but presents low therapeutic efficacy. The molecular mechanism responsible for the BAL toxicity is poorly known. In the present study, the effect of BAL and inorganic and organic mercury on Ca²⁺ transport by Ca²⁺-ATPases located in the sarco/endoplasmic reticulum of fast-skeletal muscle and brain was examined. Ca²⁺ uptake by brain and fast-skeletal muscle microsomes was inhibited in a dose-dependent manner by Hg²⁺. The calculated IC₅₀ for Ca²⁺ uptake inhibition by HgCl₂ was 1.05 ± 0.09 M (n = 8) for brain and 0.72 ± 0.06 M (n = 9) for muscle. The difference was significant at p < 0.01 (data expressed as mean ± SD). At a low concentration (1 M), 2,3-dimercaptopropanol had no effect on Ca²⁺ uptake by brain or muscle vesicles and did not abolish the inhibition caused by Hg²⁺. A high concentration of BAL (1 mM) nearly abolished the inhibition caused by 1.75 M HgCl₂ or 6 M CH₃HgCl in skeletal muscle. Surprisingly, at intermediate concentrations (40–100 M) BAL partially inhibited Ca²⁺ transport in brain but had no effect on muscle. Furthermore, ATP hydrolysis by brain or muscle microsomes was not inhibited by BAL. These results suggest that in brain microsomes BAL affects in a different way Ca²⁺ transport and ATP hydrolysis. The increase in BAL concentration observed after toxic administration of this compound to experimental animals may contribute to deregulate Ca²⁺ homoeostasis and, consequently, to the neurotoxicity of BAL.