Effect of CH3HgCl and several transition metals on the dopamine neuronal carrier; peculiar behaviour of Zn2+

CH₃Hg⁺ and metal ions inhibited the specific binding of (1-[2-(diphenylmethoxy)ethyl]-4-(3-phenyl-2-[1-³H]propenyl) piperazine) ([³H]GBR 12783) to the dopamine neuronal carrier present in membranes from rat striatum with a general rank order of potency CH₃Hg⁺ > Cu²⁺ > Cd²⁺ > Zn²⁺ > Ni²⁺ = Mn²⁺ = Co²⁺, suggesting that -SH groups are chiefly involved in this inhibition. Five millimolar dithiothreitol reversed the rather stable block of the specific binding produced by Cd²⁺ or Zn²⁺. An increase in the concentration of Na⁺, or addition of either K⁺ or Ca²⁺ reduced the inhibitory effects of metal cations, except Cu²⁺. Zn²⁺ (3 μM) reduced the inhibitory potency of Cd²⁺ on the binding but was ineffective against CH₃Hg⁺ and Cu²⁺. Zn²⁺ at 0.3 to 10 μM significantly enhanced the specific binding of [³H]GBR 12783 and [³H]cocaine by 42 to 146%. Zn²⁺ (3 μM) increased the affinity of all pure uptake inhibitors tested and of the majority of the substrates for the [³H]GBR 12783 binding site. Dissociation experiments revealed that Zn²⁺ both inhibited and enhanced the [³H]GBR 12783 binding by recognizing amino acids located close to or in the radioligand binding site. Micromolar concentrations of Zn²⁺ noncompetitively blocked the [³H]dopamine uptake but they did not modify the block of the transport provoked by pure uptake inhibitors. These findings suggest that Na⁺, K⁺, Ca²⁺ and metal ions could recognize some -SH groups located in the [³H]GBR 12783 binding site; low concentrations of Zn²⁺ could allow a protection of these -SH groups.     

The time-course of cadmium-thionein synthesis in the rat

After the intravenous injection of Cd²⁺ (1.6 mg/kg body wt) the cation accumulates to slightly greater concentrations in the liver and kidneys of the 10-week-old female Wistar rat, than in these organs of the 10-week-old male. Ionic Cd²⁺ is more toxic for the female than for the male, the ld₅₀ values being 1.4 (1.2?1.7) and 2.2 (1.9?2.6) mg Cd²⁺/kg body wt, respectively. This difference in toxicity is not correlated with differences in rates of hepatic cadmium-thionein synthesis. In both sexes, uptake of Cd²⁺ by the liver is complete within 1 hr. In the male rat there is a lag phase of 3–4 hr between the administration of Cd²⁺ and the onset of the induced synthesis of thionein in the liver. Once this synthesis occurs, the content of the metallothionein increases rapidly, Cd²⁺ being transferred to the apoprotein from proteins of high molecular weight that provide the initial binding sites for the cation in the soluble fraction of the liver. At a dose level of 1.6 mg Cd²⁺/kg, synthesis of the metallothionein is mainly complete within 8 hr and appears to be unaffected by age; both the length of the lag phase and subsequent rate of formation of the hepatic metalloprotein in the 80-week-old male rat being the same as those in the 10-week-old animal. In contrast with the male, approximately 5 per cent of the total Cd²⁺ in the soluble fraction of the liver of the female rat is bound as the metallothionein within 1 hr after the administration of the cation. This incorporation of Cd²⁺ into the metalloprotein is attributed to replacement by Cd²⁺ of Zn²⁺ in zinc-thionein, which is present in low concentration in the liver of the normal female rat. This initial replacement is followed by a slow increase in the content of thionein-bound Cd²⁺ during the following 2–3 hr. Thereafter, presumably due to stimulation by the Cd²⁺-induced messenger, the rate of synthesis increases rapidly to a maximum, which is at least equal to, and occurs at the same time, as that in the male. At 24 hr after the administration of Cd²⁺ to the male rat, the content of Zn²⁺ in the hepatic metallothionein is similar to that of Cd²⁺. Replacement of this Zn²⁺ by Cd²⁺ may account for the immediate incorporation of the latter cation into the hepatic metallothionein that occurs when the animals are given a second dose of Cd²⁺. After this initial replacement the synthesis of thionein, which has been primed by the first dose of Cd²⁺, occurs without lag on exposure to the second, and both Zn²⁺ and Cd²⁺ are incorporated into the metallothionein. Intravenous injection of 1.6 mg Cd²⁺/kg body wt also leads to a rapid accumulation of the cation in the kidney, but does not induce the synthesis of the metallothionein in this organ of either the male or female rat during the following 48 hr.     

Metabolism of cadmium in the neonate: Effect of hepatic zinc,copper and metallothionein concentrations on the uptake of cadmium in the rat liver

The accumulation and subcellular distribution of Cd²⁺ (1 mg/kg body wt, i.p.) in the liver of the neonatal rat is age-dependent. At 4 hr after treatment the liver Cd²⁺ contents in the 12-day-old, 20-day-old and adult rat are similar and greater than in the 2-day-old animal. The differences in hepatic Cd²⁺ concentration in the older age groups are consistent with the nonlinear weight gain of the liver in the developing animal. In the hepatic cytosol Cd²⁺ is incorporated into a high molecular weight and metallothionein fractions and transferred from the former to the latter. This process occurs less rapidly with increasing age. The uptake of Cd²⁺ by the whole liver and the hepatic metallothionein is not related to the total liver concentration of Zn²⁺ or copper and is not significantly influenced by the concentration of pre-existing metallothionein or the concentration of thionein-bound Zn²⁺ or copper. The results are discussed in relation to the possible effects of Cd²⁺ on the liver metabolism and tissue distribution of Zn²⁺ and copper in the developing animal.     

Binding of cadmium ions by rat liver and kidney

Most of the bound Cd²⁺, which accumulates in the livers and kidneys of adult rats after the subcutaneous injection of CdCl₂ (2·2 μmoles/100 g body wt) is recovered as a single, heat-stable fraction from the soluble components of these tissues. Although this fraction in either organ also binds Zn²⁺, Cd²⁺ does not displace Zn²⁺ from any of the normal, soluble Zn²⁺-metalloproteins.     

The synthesis of metallothionein and cellular adaptation to metal toxicity in primary rat kidney epithelial cell cultures

Single cells were isolated from rat (100–150 g) kidney cortex slices by mild trypsinization. They were plated on plastic culture dishes and were maintained in a selective Eagle's D-Valine Minimum Essential Media (MEM) to form an epithelial cell monolayer culture. The fibroblast growth was retarded by D-Valine. The cells in monolayer culture accumulated cadmium when they were incubated with a ¹⁰⁹CdCl₂ (10^?5 M) containing medium. The synthesis of metallothionein by these cells was demonstrated by incorporation of [³⁵S]cysteine and ¹⁰⁹CdCl₂ into a heat stable protein (mol. wt 10 000) within 8 h of exposure and also by immunoprecipitation using a specific antibody to rat liver metallothionein. The cytotoxic effects of Zn²⁺, Cu²⁺, Cd²⁺ and Hg²⁺ were studied in culture after addition of various concentrations of metal salts (10^?5 –10^?2 M). Disrupted cellular colonies with severe cell damage were observed after addition of 10^?3 M Cd²⁺ or Hg²⁺ as CdCl₂ or HgCl₂ while similar toxicity was observed only after addition of 10^?2 M Zn²⁺ or Cu²⁺ as ZnSO₄ or CuSO₄ to confluent cell cultures. The cellular damage due to Cd²⁺ was protected when the cells were pretreated with Cd²⁺ (10^?5 M) for 24 h and these cells could tolerate much higher concentrations of cadmium (10^?2 M). These results indicate a direct protective role of intracellular metallothionein in the cellular toxicity of cadmium.     

Induction of the Conversion of Xanthine Dehydrogenase to Oxidase in Rabbit Liver by Cu2+, Zn2+ and Selenium Ions

Abstract— Effects of Cu²⁺, Zn²⁺, Fe²⁺ and selenium ions on the conversion of xanthine dehydrogenase to oxidase in rabbit liver were examined. Under basal conditions, xanthine oxidase activity represented only 16% of the total xanthine oxidase plus dehydrogenase activity. Cu²⁺ (2–10 μm ), Zn²⁺ (5–30 μm ) and selenium ions (5–100 μm ) brought about the conversion of xanthine dehydrogenase to oxidase in a dose-dependent manner. The concentrations of Cu²⁺, Zn²⁺ and selenium ions required for increasing xanthine oxidase activity by 50% was approximately 4, 10 and 20 μm , respectively. On the other hand, Fe²⁺ had no effect on the conversion of the enzyme up to 100 μm . These results suggest that Cu²⁺, Zn²⁺ and selenium ions have the potential to modulate the conversion of xanthine dehydrogenase to oxidase in rabbit liver.     

Effects od divalent cations on neuromuscular transmission in the chick

In comparative studies on the effects of nine divalent cations (Mn²⁺, Co²⁺, Ni²⁺, Cd²⁺, Zn²⁺, Sr²⁺, Cu²⁺, Ba²⁺ and UO₂²⁺ on twitch responses to indirect and direct stimulations, on acetylcholine response and on Ca²⁺ uptake, it was found that Cd²⁺ was the most potent in inhibiting transmitter release from the motor nerve terminals but was without appreciable effect on the chick biventer cervicis muscle. Mn²⁺, Co²⁺, Ni²⁺ and Zn²⁺ were less potent than Cd²⁺ in producing inhibition at presynaptic sites but were morep potent at postsynaptic sites. By contrast, UO₂²⁺ potentiated twitch responses to indirect stimulation and induced a contracture of the muscle in the presence of physostigmine. Cu²⁺ and Ba²⁺ were particularly potent in inducing a contracture and at higher concentrations abolished twitch responses to indirect and direct stimulation. Sr²⁺ at the very high concentration of 22 mM had only a week inhibitory action on twitch responses but was the only cation capable of substituting for Ca²⁺ in inducing contraction. The studies on the effects of these cations on denervated muscle also led to the conclusion that Cd²⁺ and UO₂²⁺ act mainly on presynaptic nerve terminals and that Mn²⁺, Co²⁺, Ni²⁺ and Zn²⁺ act on presynaptic sites preferentially to postsynaptic sites while Cu²⁺ and Ba²⁺ act mainly on muscle. The effects of these cations can be antagonized by high Ca²⁺ (10 mM) but those of Cu²⁺ cannot. Cysteine (2.5p mM) antagonized the effects of all the cations except Mn²⁺, Ba²⁺ and UO₂²⁺. d-Tubocurarine suppressed the contracture induced by UO₂²⁺ but not that induced by Cu²⁺ and Ba²⁺. These cations affected ⁴⁵Ca²⁺ uptake and ionic contents of the muscle differently. ⁴⁵Ca²⁺ uptake of the muscle was decreased by Mn²⁺, Co²⁺ and Ni²⁺, not altered by UO₂²⁺ but increased by Cd²⁺, Zn²⁺, Sr²⁺, Cu²⁺ and Ba²⁺. In addition to greatly increasing ⁴⁵Ca²⁺ uptake, Cu²⁺ also increased calcium and sodium but decreased the potassium content of muscle. Zn²⁺ and Sr²⁺ increasedp the calcium content slightly but the other cations did not effect ionic contents appreciably. It is concluded that these cations exert their effects on nerve or on muscle depending on the ligands of the ligands of the membrane bindingp these cations. Some of the cations bi-nd with the—SH group of the membrane and thus alter the transmembrane movement of Ca²⁺ bu the others do not.     

Effect of cadmium on the extracellular Na+, K+, and Ca2+ in the gill and small intestine of Goldfish Carassius auratus

In this study, the toxic effect of cadmium on extracellular Na⁺, K⁺, and Ca²⁺ in the gill and small intestine of goldfish Carassius auratus was determined with the technique of ion chromatograph. Two-way ANOVA indicated that the two factors (Cd²⁺ treatment and time) and the interaction factor had significant effect on the level of Na⁺, K⁺, and Ca²⁺ in the small intestine and gill. 1.0 mg/L Cd²⁺ significantly increased Ca²⁺ level in the small intestine, but Ca²⁺ level in the gill was significantly decreased by 1.0 and 5.0 mg/L Cd²⁺ at 24, 48, and 72 h. Na⁺ and K⁺ level in the small intestine and gill was increased by 1.0 mg/L Cd²⁺ at three time points, but increased by 5.0 mg/L Cd²⁺ at a certain different time. In addition, Na⁺ level was significantly decreased by 5.0 mg/L Cd²⁺ at 24 or 48 h in the small intestine and gill. The results indicated that Cd²⁺ played an important role in regulating the level of Na⁺, K⁺, and Ca²⁺ in the small intestine and gill of goldfish C. auratus. A method was constructed to investigate the extracellular Na⁺, K⁺ and Ca²⁺ in the tissues of gold fish with ion chromatography.     

Effect of Heavy Metals on Dopamine,Noradrenaline and Serotonin Uptake and Release in Rat Brain Synaptosomes

Abstract: The effects of some heavy metals on the initial high affinity uptake and spontaneous release of tritiated dopamine (³H-DA), noradrenaline (³H-NA) and 5-hydroxytryptamine (³H-5-HT) were studied in vitro in rat striatal, cortical and hypothalamic synaptosomes, respectively. As uptake inhibitors, metals were quite inactive in these conditions. At 10 μM Cu²⁺ was most potent, inhibiting ³H-DA and ³H-5-HT uptake nearly completely while inhibition of ³H-NA uptake varied. ³H-DA uptake was in addition inhibited slightly by Zn²⁺, sometimes by Sn²⁺ but never by Co²⁺, Hg²⁺ or Mn²⁺. Unexpectedly Pb²⁺ and Cd²⁺ tended to increase the synaptosomal ³H-DA uptake. ³H-5-HT uptake was affected least while that of ³H-NA showed some diversity. Zn²⁺, Pb²⁺ and Sn²⁺ induced inhibition of ³H-NA uptake possibly by direct interference with ³H-NA. As to the spontaneous release of tritiated amines during short incubation from preloaded synaptosomes, Cd²⁺ decreased that of ³H-DA at high concentrations but Hg²⁺, Pb²⁺, Sn²⁺ and Zn²⁺ were ineffective. The results suggest that in vitro the uptake and the release of ³H-DA are more affected than those of other amines. The inhibitory mechanisms of monoamine uptake may include both direct effects on synaptosomes and indirect ones by interference with the amines themselves.     

Metals (Ag+, Cd2+, Cr6+) affect ATPase activity in the gill,kidney, and muscle of freshwater fish Oreochromis niloticus following acute and chronic exposures

Freshwater fish Oreochromis niloticus were individually acutely exposed to different concentrations (0, 0.1, 0.5, 1.0, and 1.5 μg/mL) of Cd²⁺, Cr⁶⁺, and Ag⁺ for 96 h and 0.05 μg/mL concentration of the same metals for different periods (0, 5, 10, 20, and 30 days) chronically. Following each experimental protocol, Na⁺/K⁺‐ATPase, Mg²⁺‐ATPase, and Ca²⁺‐ATPase activities were measured in the gill, kidney, and muscle of O. niloticus. In vitro experiments were also performed to determine the direct effects of metal ions (0, 0.1, 0.5, 1.0, and 1.5 μg/mL) on ATPases. Except Ag⁺, none of the metals caused fish mortality within 30 days. Silver killed all the fishes within 16 days. Metal exposures generally decreased Na⁺/K⁺‐ATPase and Ca²⁺‐ATPase activities in the tissues of O. niloticus, although there were some fluctuations in Mg²⁺‐ATPase activity. Ag⁺ and Cd²⁺ were found to be more toxic to ATPase activities than Cr⁶⁺. It was also observed that metal efficiency was higher in the gill than in the other tissues. Results indicated that the response of ATPases varied depending on metals, exposure types, and tissues. Because ATPases are sensitive to metal toxicity, their activity can give valuable data about fish physiology. Therefore, they may be used as a sensitive biomarker in environmental monitoring in contaminated waters. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 707–717, 2013.     

Cadmium resistance and content of cadmium-binding protein in two enzyme-deficient mutants of mouse fibroblasts (L-cells)

The toxic Cd²⁺ ion accumulates in mammalian organisms, the main storage organs are apparently the liver and the kidney. In these organs Cd²⁺ is bound to low molecular weight proteins (thioneins) as metallothionein. We describe here the development of resistance to otherwise lethal concentrations of Cd²⁺ by two non-epithelial cell lines, both derived from mouse fibroblasts (L-cells). One of the cell lines (clone ID) is deficient in thymidine kinase and resistant to 5-bromodeoxyuridine, the other (A9) deficient in hypoxanthine-guanine phosphoribosyl transferase and resistant to 8-azaguanine. After stepwise increase in Cd²⁺ concentration, clone 1D cells had apparently normal growth rate in the presence of 100 micromolar Cd²⁺ after 6 months of Cd treatment. The A9 cells were apparently more sensitive to Cd²⁺, after about one year's Cd treatment they had apparently normal growth in the presence of 100 micromolar Cd²⁺. This concentration of Cd²⁺ would kill cells of both cell lines not previously exposed to Cd. In the resistant A9 cells about 40 per cent of the cadmium were bound to a cadmiumbinding protein (Cd-BP) of molecular weight of about 12,000, most probably metallothionein, in the resistant clone 1D cells the corresponding figure was 60 per cent. The non-resistant cell lines had apparently no metallothionein. We have thus found that also non-epithelial cells can synthesize low molecular weight Cd-BP and that there apparently is a good correlation between cadmium resistance and content of Cd-BP.     

Mechanism of manganese-induced tolerance to cadmium lethality and hepatotoxicity

Pretreatment with Mn²⁺ is known to produce tolerance to Cd²⁺-induced lethality. This study was designed to determine the mechanism of tolerance to Cd²⁺-induced lethality and hepatotoxicity following Mn²⁺ pretreatment. Rats given 36 μmoles Cd²⁺/kg, i.v., died within 10–20 hr while only one of nine rats pretreated with Mn²⁺ (250 μmoles/kg, s.c., 48 and 24 hr prior to Cd²⁺ challenge) died. Ten hours after Cd²⁺, plasma aspartate aminotransferase and sorbitol dehydrogenase activities were elevated markedly, and extensive histopathologic lesions of the liver were evident in control rats but not in Mn²⁺- pretreated rats. To examine the mechanism of this tolerance, distribution of Cd²⁺ to fourteen organs and the subcellular distribution in six organs were determined in control and Mn²⁺-pretreated rats. Two hours after challenge (31 μmoles Cd²⁺/kg, i.v., 0.75 μCi ¹⁰⁹Cd²⁺/μmole Cd²⁺), the distribution of Cd²⁺ to liver markedly increased after Mn²⁺ pretreatment with concomitant decreases in other tissues. Mn²⁺ pretreatment also resulted in a marked difference in the hepatic subcellular distribution of Cd²⁺ with more present in cytosol and less associated with organelles. Gel-filtration chromatography indicated that most cytosolic Cd²⁺ was bound to a low molecular weight protein. Isolation and partial characterization of this protein suggest that it is identical to metallothionein (MT); it had a similar relative elution following gel-filtration chromatography, had low absorbance at 280 nm and, after separation into two isoproteins by DEAE A-25 anion exchange chromatography, had the same mobility after electrophoresis on non-denaturing polyacrylamide gels as Cd²⁺-induced metallothioneins. These data suggest that Mn²⁺ pretreatment reduces Cd²⁺-induced hepatotoxicity by altering the hepatic subcellular distribution of Cd²⁺ with more Cd²⁺ binding to MT in the cytosol. This decreased hepatotoxicity is probably responsible for tolerance to Cd²⁺-induced lethality.     

Studies on the reaction catalyzed by transport (Na, K) adenosine triphosphatase. I. Effects of divalent metals

The effects of divalent metals (Cu²⁺, Zn²⁺, Fe²⁺ and Pb²⁺) on a microsomal preparation of NaK-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from beef cerebral cortex were studied. These metals are all potent inhibitors of the enzyme with I₅₀ values of 1 μM for Cu²⁺ and Zn²⁺, 3 μM for Fe²⁺ and 20 μM for Pb²⁺. Kinetic studies examining the effect of low concentrations of divalent metals on K_m and V for MgATP are reported. The results indicate that Fe²⁺ and Pb²⁺ are competitive inhibitors of NaK-ATPase with K_i values of 1.60 μM and 1.90 μM respectively. Cu²⁺ and Zn²⁺ are noncompetitive inhibitors of NaK-ATPase with K_i values of 1.18 μM and 3.48 μM respectively.     

Effects of Cd2+, Pb2+ and CH3Hg+ on high voltage-activated calcium currents in pheochromocytoma (PC12) cells: potency,reversibility, interactions with extracellular Ca2+ and mechanisms of block

Effects of the neurotoxic heavy metals Cd²⁺, Pb²⁺ and CH₃Hg⁺ on current carried by Ca²⁺ ions (I_Ca) through high-voltage activated Ca²⁺ channels in nerve growth factor (NGF)-differentiated pheochromocytoma (PC12) cells were examined to characterize possible differences in the mechanism of action of these metals on Ca²⁺ channel function. Specifically, the potency and reversibility of effect on I_Ca by each metal was examined, as well as the relationship between extracellular [Ca²⁺] and potency of block of I_Ca by Cd²⁺ and Pb²⁺. In addition, the effect of each of these metals on Ca²⁺ channels when applied to the intracellular side of the membrane was also examined. When extracellular solution contained 20, 10 or 5 mM Ca²⁺, the estimated IC₅₀ values (total metal concentration) for block of I_Ca were 15, 10, and 6.5 μM for Cd²⁺ and 7.5, 2.0 and 1.1 μM for Pb²⁺, respectively. CH₃Hg⁺ (1–10 μM) blocked I_Ca (20 mM Ca²⁺) in a time- and concentration-dependent manner. When cells were washed with metal-free solutions, block of I_Ca by Cd²⁺ was reversed rapidly, whereas block by Pb²⁺ was reversed only partially, and block of I_Ca by CH₃Hg⁺ was not reversed. When Pb²⁺ and CH₃Hg⁺ treated cells were washed in metal-free solutions containing 50 μM d-penicillamine (DPEN), block of I_Ca by 10 μM Pb²⁺ was rapidly and completely reversed, whereas, block of I_Ca by 5 μM CH₃Hg⁺ was not reversed. Higher concentrations (500 μM) of 2,3-dimercapto-1-propane sulfonic acid (DMPS) did reverse partially the block of I_Ca by 5 and 10 μM CH₃Hg⁺. When Cd²⁺, Pb²⁺ or CH₃Hg⁺ was present in the intracellular solution, Ca²⁺ channel currents were significantly reduced. These results characterize effects of Cd²⁺ on Ca²⁺ channels and demonstrate that Cd²⁺, Pb²⁺ and CH₃Hg⁺ differ in their actions on Ca²⁺ channels.     

Cadmium/zinc-metallothionein induces DNA strand breaks in vitro

The in vitro DNA strand breaking activity of metallothionein (MT) containing Cd²⁺ and Zn²⁺ in a molar ratio of 52 is described. Studies with radical scavengers and electron paramagnetic resonance spectroscopy indicate that the DNA damage might be caused by a radical species formed by the native protein (i.e., MT) charged with the heavy metal ions. No DNA strand breaks are detectable with the heat-denatured MT or with Cd²⁺ or Zn²⁺ alone. Inhibition studies using EDTA as a metal ion chelator orN-ethylmaleimide to alkylate sulfhydryl groups suggest that both the bound heavy metal ions as well as the SH groups of the various cysteine residues of MT may be involved in the MT-dependent DNA cleavage. Further characterization showed that the DNA cleavage is more likely random than sequence- or base-specific. These observations may provide a clue in the search for initial events in Cd-related carcinogenicity.     

Relationships between subcellular distributions of cadmium and perturbations in reproduction in the polychaete Neanthes arenaceodentata

The accumulation and subcellular distribution of Cd in the polychaete worm, Neanthes arenaceodentata, were examined following an eleven-week period of exposure to a range of free cadmium ion activities, [Cd²⁺]. The accumulation of Cd in N. arenaceodentata was directly proportional to [Cd²⁺] in seawater at lower concentrations (10⁻¹² M to 10⁻¹⁰ M) but deviated from proportionality at higher concentrations (10⁻⁹ M and 10⁻⁸ M). This deviation in proportionality at higher [Cd²⁺] was attributable to a relative increase in the concentration of metal associated with the metallothionein and the very low molecular weight metal-ligand pools of the cytosol.Reproductive potential was also monitored in these organisms to examine the ecological significance of Cd accumulation and shifts in its subcellular distribution. Perturbations in reproduction were observed at 10⁻⁸ M [Cd²⁺] which coincided with the deviation in proportionality of Cd accumulation and increased accumulation of Cd in the cytosol.     

Effects of Divalent Cations on Adenosine Agonist Binding to A1 Receptors and Non-A1/Non-A2 Sites in Rat Cerebral Cortex

Abstract: In the present study results are reported concerning the effects of several divalent cations on the binding characteristics of [³H]-cyclohexyladenosine on A1 adenosine receptors and of [³H]-N-ethylcarboxamidoadenosine on non-A1/non-A2 sites in membranes from cerebral cortex of the rat. The [³H]-cyclohexyladenosine binding to A1 receptors was dose-dependently increased by Mn²⁺, Co²⁺, Ca²⁺. The binding characteristics of the agonist were differently affected by Ca²⁺/Mn²⁺ and Mg²⁺. Ca² and Mn²⁺ increased the B_max value without any change in K_d, whereas Mg²⁺ decreased the K_d value without changing the B_max. In the presence of Ca²⁺ and Mg²⁺ the K_d value was similar to that obtained in the presence of Mg²⁺, whereas the B_max value was similar to the apparent number of binding sites calculated in the presence of Ca²⁺. The cations, Cu²⁺, Cd²⁺, Zn²⁺, decreased the Al binding with IC₅₀, values of 19.6 μM, 39.2 μM and 103.9 μM, respectively. The binding characteristics of [³H]-N-ethylcarboxamidoadenosine to non-A1/non-A2 sites were affected by Ca²⁺, Mn²⁺, Co²⁺ and Mg²⁺ in the opposite manner to A1 receptors. They decreased the binding with IC₅₀ values of 20.1 mM, 22.8 mM, 93.0 mM and 18.1 mM, respectively. This occurs through an enhancement in K_d, values without changes in the number of binding sites. The findings on A1 receptor and non-A1/non-A2 binding site, taken together, suggest that cations could also exert a modulatory action via specific interactions with divalent cation binding sites on the receptor molecule.     

The lysosome among targets of metformin: new anti-inflammatory uses for an old drug?

Background: Rheumatoid arthritis and type-2 diabetes exhibit progressive co-morbidity. Chloroquine (CQ) reportedly improves both. CQ inhibits lysosomal function in cultured cells at supra-therapeutic concentration; however, this is doubted as target mechanism. Some anti-diabetic biguanides are metal-interactive lysosomal inhibitors; and all bind Zn²⁺.

Objectives: i) To bioassay the potency of CQ using ³H-leucine release from perfused myocardial tissue. ii) To determine whether metformin (MET) is CQ-mimetic, and interactive with Zn²⁺.

Results: Therapeutic CQ concentration (0.1 – 0.5 μM) clearly does cause lysosomal inhibition although delayed and submaximal. MET alone (10 μM) caused sub-maximal inhibition. Supra-physiological extracellular Zn²⁺ (5 – 50 μM) alone increased tissue Zn²⁺ content, and inhibited lysosomal proteolysis. Physiological equivalent Zn²⁺ (approximately 1 μM) had no effect. MET (≤ 25 μM) and Zn²⁺ (≤ 1 μM) exhibited astounding 10 – 100 fold anti-lysosomal synergy. Cathepsin B was 50% inhibited by 1 μM Zn²⁺, and is reportedly inhibited by gold agents.

Interpretation: MET somehow increases the natural inhibitory action of action of Zn²⁺ against cysteinyl proteases. TNF-alpha activates lysosomal function; and CatB is among post-receptor players. MET might decrease antigen processing in specialized cells, and lysosomal hyper-catabolism in other cells.

Conclusions: Trials of MET for new use as an anti-inflammatory agent are suggested. Guanidylguanidine is a practical pharmacophore for synthesis of future anti-lysosomal agents.     

Changes in the contractile responses to carbachol and in the inhibitory effects of verapamil and nitrendipine on isolated smooth muscle preparations from rats subchronically exposed to Co2+ and Ni2+

Male Wistar rats were exposed to subtoxic doses of Co²⁺ or Ni²⁺, receiving Co(NO₃)₂ or NiSO₄ with drinking water for 30 days. No significant differences in the body weight and no visible changes in the behaviour of the controls and experimental animals were established. Cumulative concentration-effect curves for carbachol were obtained in ileum and trachea isolated from control and Co²⁺- or Ni²⁺-treated rats. The effect of the Ca²⁺ antagonists on the carbachol-induced contractions was studied by adding increasing concentrations of verapamil or nitrendipine to the bath solution 20 min prior to carbachol. The results showed that exposure of rats to subtoxic doses of Co(NO₃)₂ or NiSO₄ altered the contractile responses to carbachol. The changes in the pD₂ values and the shift to the left of the concentration-effect curves suggest a higher sensitivity to carbachol in preparations from the ileum of Co²⁺- or Ni²⁺-exposed rats. The tracheal strips isolated from control and heavy metal-treated rats showed a less potent sensitiveness to carbachol as compared to the ileal segments. An opposite tendency for decreased cholinergic reactivity was observed in tracheal strips from Co²⁺- and Ni²⁺-treated animals. The inhibitory effect of the Ca²⁺-antagonists on the contractility of ileal preparations from Co²⁺-treated rats increased at all concentrations of verapamil and at the highest concentration of nitrendipine, but decreased at lower concentrations of nitrendipine. The effect of verapamil on the preparations from Ni²⁺-exposed rats was unchanged or even decreased at higher verapamil concentrations. The inhibitory effect of nitrendipine on preparations from Ni²⁺-exposed rats decreased at the lowest concentration but increased at the highest concentration of the blocker. In Co²⁺- or Ni²⁺-treated tracheal preparations verapamil inhibited the contractions induced by low and medium concentrations of carbachol but increased the maximal contractile responses to high concentrations of carbachol.     

An investigation of the role of metallothioneins in protection against the acute toxicity of the cadmium ion.

Pretreatment of female rats with a low dose of Cd²⁺ protects them against a subsequent, normally lethal dose of the same cation, and also induces the synthesis of hepatic cadmium-thionein. This protection, however, is maximal 1–3 days after pretreatment and then decreases, whereas both the increased content, and capacity for the synthesis of the metallothionein are maintained. Because of this increased capacity for cadmium-thionein synthesis a greater percentage of a subsequent dose of Cd²⁺ is retained in the liver of the pretreated animal, than in the non-pretreated control. Uptake of the cation into other organs (i.e. heart. kidney, pancreas and spleen), however, is unaffected by the pretreatment.Although Cd²⁺ is bound more strongly by the apoprotein, thionein, than is Zn²⁺. pre-induction of hepatic zinc- thionein by restriction of food intake, does not lead to increased resistance to the toxic cation.These observations suggest that pre-induced metallothioneins do not have a significant role in the protection against the acute toxicity of Cd²⁺.