microM Zinc Research Articles

Zinc inhibits Ca2+ transport by rat brain NA+/Ca2+ exchanger.

Calcium transport by the Na+/Ca2+ exchanger was measured in plasma membranes vesicles purified from rat brain and in primary rat cortical cell culture. Sodium-loaded vesicles rapidly accumulate Ca2+ via Na+/Ca2+ exchange (Na+(i)-dependent Ca2+ uptake). Extravesicular zinc inhibited Na+/Ca2+ exchange as evidenced by a reduction of the initial velocity of Ca2+ uptake. Significant inhibition of Ca2+ uptake was seen at concentrations of zinc as low as 3 microM. Lineweaver-Burk analysis of the data was consistent with noncompetitive inhibition with respect to extravesicular Ca2+ concentration. The Ki for zinc inhibition of Ca2+ uptake determined from a Dixon plot was 14.5 microM. This is within the range of zinc concentrations thought to be obtained extracellularly after excitation. When vesicles were preloaded with Ca2+, extravesicular zinc also inhibited reversal of Na+/Ca2+ exchange (Na+(i)-dependent Ca2+ release) although its potency was much less: concentrations of > or = 30 microM zinc were required. Zinc inhibition of Ca2+ release was not Na+ dependent. Na+(i)-dependent calcium uptake by rat cortical cells in primary culture also was inhibited by zinc. The extent of inhibition was similar to that seen for inhibition of Na+(i)-dependent Ca2+ uptake in membrane vesicles, but the potency was less. The results suggest that Ca2+ transport by the Na+/Ca2+ exchanger is inhibited by concentrations of zinc thought to be attained extracellularly after excitation.

Characterization of a multi-copper enzyme, nitrous oxide reductase, from Rhodobacter sphaeroides f. sp. denitrificans.

The nitrous oxide reductase from the photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans, has been purified. The enzyme is composed of two identical subunits of 66 kDa, and contains four copper atoms per subunit. Copper supplementation of the medium resulted in a 3.5-fold increase in the enzyme yield with doubly enhanced specific activity. The activity of the purified nitrous oxide reductase was completely inhibited by 100 microM zinc ions.

The effect of zinc ions on the activity of metallo-beta-lactamases of Bacteroides fragilis.

The zinc supplement required to achieve the maximum activity of metallo-beta-lactamases from 12 Bacteroides fragilis isolates was investigated. Changes in absorbance of imipenem in a spectrophotometric assay with crude cell extracts were performed in the presence and absence of various concentrations of zinc sulphate. The greatest degree of imipenem hydrolysis was seen with the addition of between 50 and 500 microM zinc sulphate, and the degree of stimulation of enzyme activity in the strains tested varied six-fold. Increasing the zinc sulphate concentration resulted in inhibition of hydrolysis with extracts of low enzymic activity. These findings indicate the importance of determining the optimal zinc concentration for each strain tested in kinetic studies of metallo-beta-lactamases.

A reappraisal of the role of zinc in life and death decisions of cells.

There is a great deal of interest in chemicals and biochemicals that can modulate apoptosis. As will be discussed, zinc, an essential trace element, can induce as well as block apoptosis. High concentrations of extracellular zinc (500-1000 microM) have frequently been used to block apoptosis or programmed cell death in a variety of systems. Early investigators provided evidence that this concentration of zinc could block DNA fragmentation that is often associated with apoptosis. Since zinc plays a role in many aspects of cell function, there are probably many sites in a death pathway that zinc could potentially modulate. In the case of glucocorticoid-mediated apoptotic death, new evidence presented herein indicates that high zinc can also block the binding of steroids to the glucocorticoid receptor thereby inhibiting the death signal itself. In this case, zinc probably binds to the vicinal cysteines in the receptor ligand binding site thereby blocking binding of glucocorticoid. Indeed, glucocorticoid-induced apoptosis in thymocytes has become one of the most frequently studied systems and is a focal point of this review. Studies herein will show that unlike zinc other trace-like metals such as nickel, copper, cadmium, and gold do not afford thymocytes protection against the DNA fragmentation induced by glucocorticoid-mediated cell death. Interestingly, in attempting to determine if lower or more physiological concentrations of zinc could provide protection against apoptosis, it was found that 80-200 microM zinc could actually induce death in 40% of CD4+ CD8+ alpha beta TCR10CD3(10) thymocytes. From these experiments one might have been optimistic that zinc could, indeed, be a modulator of cell death. However, this thought has been overshadowed by growing evidence that zinc does not provide long-term protection to so-called surviving cells.

Zinc attenuation of GDP binding to brown adipocytes mitochondria in genetically obese (ob/ob) mice.

In this study, we investigate the in vitro effect of zinc addition on guanosine diphosphate (GDP) binding to mitochondria in brown adipocytes of genetically obese (ob/ob) mice. Interscapular brown adipocytes of male mice (obese; lean) at 4 and 12 wk of age were incubated with 0, 50, 100, or 200 microM zinc sulfate. Mitochondria were then isolated and their GDP binding capacities were measured. The GDP-binding capacities of ob/ob mice were lower than lean mice, with or without zinc addition, in both age groups (p < 0.05). Zinc addition did not have any significant effect on GDP binding in lean mice. GDP binding decreased with increasing zinc addition in ob/ob mice, and this attenuation was more predominant in 12-wk old ob/ob mice. Moreover, we found that high magnesium addition (5 mM) increased GDP binding in lean mice, but this effect was not significant in ob/ob mice. This study reveals that brown adipose tissue thermogenesis in ob/ob mice could be greatly attenuated by zinc addition, suggesting that zinc may play a regulatory role in obesity.

Zinc enhances ionic currents induced in skate Müller (glial) cells by the inhibitory neurotransmitter GABA.

We describe here a novel effect of zinc on GABA receptors of glial cells in the skate retina. The GABA-induced currents of skate Müller cells, the radial glia of the retina, are mediated by activation of GABA(A) receptors (GABAARS). In other parts of the nervous system, GABz*-)ediated currents are inhibited by zinc. However, in isolated, voltage-clamped Müller cells, coapplication of zinc (10 microM) and GABA (1 microM) resulted in enhancement of the GABA(A)R current. Surprisingly, zinc alone induced a current similar in many respects to that elicited by GABA, i.e. the reversal potential was the same as for the GABA-induced current, the current was blocked by bicuculline and picrotoxin, and the current-voltage relation obtained in the presence of 10 microM zinc was virtually identical to that obtained with 1 microM GABA. Both bicuculline and picrotoxin suppressed a current that was present with cells bathed only in Ringer, suggesting that some of the GABA channels were spontaneously open in the absence of externally applied GABA. This possibility was supported by cell-attached patch recordings. Under conditions in which potassium and calcium currents were suppressed, spontaneous channel activity was observed. Moreover, the frequency of these channel openings was greater when zinc was included in the pipette solution, and reduced when bicuculline was added. These findings suggest that zinc acts directly to enhance the GABA(A) receptor activity of the Müller cells, and raise the possibility that the subunit composition of the GABA(A)Rs of skate Müller cells differs from that of GABA(A)Rs identified previously in other neuronal and glial preparations.

Histidine‐rich glycoprotein binding to T‐cell lines and its effect on T‐cell substratum adhesion is strongly potentiated by zinc

Histidine-rich glycoprotein (HRG), a plasma protein that binds heparin and divalent cations, has been implicated in immune regulation through its ability to modulate complement function, macrophage Fc receptor expression and phagocytosis, and its ability to inhibit the proliferation of human peripheral blood T cells in vitro. In the present work we used fluorescence flow cytometry to study the binding of human HRG to the human T-cell lines Jurkat and MT4, and to the murine antigen-specific T-cell clone D10, and to study the effect of divalent cations zinc and copper on this binding. Our results show that HRG binds strongly to these cell lines at 4 degrees, and that the binding is markedly potentiated by physiological concentrations of zinc (20 microM), and to a lesser extent by copper (10 microM). In contrast to previous studies, HRG binding was largely inhibited by 50 micrograms/ml heparin, both in the absence and in the presence of zinc, suggesting that HRG interacts primarily through glycosaminoglycans on the T-cell surface. Studies using confocal fluorescence microscopy indicated that following incubation of MT4 cells with HRG in the presence of zinc at 4 degrees, the HRG was localized exclusively at the plasma membrane, but was actively internalized after incubation at 37 degrees. Interestingly, HRG interfered with the ability of D10 cells to adhere to tissue culture plastic, as well as to laminin-, collagen- or fibronectin-coated culture dishes. This effect was markedly potentiated by 20 microM zinc, and was partially reversed by heparin. The results suggest that zinc markedly potentiates the binding of HRG to T cells, and that HRG and zinc may play an important role in regulating the adhesion of T cells to other cells and the extracellular matrix.

The role of zinc in solutions used for cold preservation of pancreatic islets.

Advances in the field of organ transplantation have been made possible by progress in cold preservation as well as advances in immunosuppression, postoperative surgical care, and refinement of surgical techniques. The optimal solution for cold storage of pancreatic islets has not been defined, although recent studies have suggested that University of Wisconsin (UW) solution or derivatives may be significant advances. Zinc has a central role in the storage, synthesis, and secretion of insulin and is taken up preferentially by isolated islets to levels 30-fold higher than those of extracellular zinc. Since the currently used cold storage solutions do not contain zinc, we have investigated the effect of adding zinc to Hanks and UW solution and assessed the viability status of the islets at 0 h and at 1, 3, and 6 days. Islets of standard diameter were suspended in four solutions: Hanks solution, Hanks +20 microM zinc, UW solution, and UW +20 microM zinc. Viability was assessed by supravital staining, and each islet was scored at 0 h and at 1, 3, and 6 days. The results showed that UW solution was superior to Hanks solution for the preservation of pancreatic islets up to 6 days, but the addition of zinc was not beneficial. Despite the integral role of zinc in islet metabolism, we were unable to find a beneficial role for zinc in cold storage solutions for the purposes of islet preservation.

Induction of hepatoma cell apoptosis by c-myc requires zinc and occurs in the absence of DNA fragmentation.

Since c-myc expression is increased during apoptosis in toxin-induced liver injury in vivo, the role of c-myc in liver cell apoptosis was investigated. The human hepatoma cell line HuH-7, which constitutively expresses c-myc, was stably transfected with sense and antisense c-myc expression vectors under the control of the zinc-inducible metallothionein promoter. None of the three cell types (wild-type, sense c-myc, or antisense c-myc) underwent apoptosis when cultured in serum-free medium (SFM). With the addition of SFM plus 37.5 microM zinc, wild-type and sense c-myc-expressing cells underwent rapid cell death, whereas antisense c-myc-expressing cells exhibited increased survival. This cell death had the light, fluorescent, and electron microscopic appearance of apoptosis, but did not result in DNA fragmentation. This apoptosis could be terminated by the addition of medium containing 2% fetal calf serum or the overexpression of bcl-2 but not by supplementation with specific growth factors. Altering c-myc expression did not affect cellular metallothionein mRNA levels or the rate of cell death from copper or cadmium. The requirement for zinc and absence of DNA fragmentation in c-myc-induced hepatoma cell apoptosis under serum-free conditions provides further evidence of the complex regulation of apoptosis in different cell types.

Influence of cAMP-effector-agonists on the synthesis of metallothionein in rat primary hepatocytes

The model of rat primary hepatocytes incubated in DMEM/F12 (Ham) medium was used for studying the influence of the cAMP-effectors epinephrine (100 microM), norepinephrine (100 microM), glucagon (1 microM) and isoproterenol (1-1000 microM) as well as the synthetic cAMP-analogon dibutyryl-cAMP on the metabolism of metallothionein. Liver parenchymal cells isolated by a two-step collagenase perfusion were incubated with DMEM/F12 containing 5% (v/v) fetal calf serum (FCS) and 20 microM zinc in Petri dishes. Experiments were initiated after a 24 h equilibration period by adding the agonists for 18 h. MT in hepatocyte homogenates was quantified by the 109Cd-hemoglobin-binding assay. Cell viability was assessed by the activity of the cytosolic enzyme lactate dehydrogenase (LDH) liberated into the culture medium and by trypan blue exclusion. Isoproterenol and glucagon produced a significant increase of cytosolic MT about 50%. In contrast, incubation with epinephrine and norepinephrine did not lead to any significant effects in the amount of hepatic metallothionein. Simulating the influence of cAMP by dibutyryl-cAMP (500 microM) did not affect the content of hepatic metallothionein. To examine transcriptional and translational regulatory effects supplementation of cycloheximide (0.1-500 microM) and actinomycin D (0.1-100 microM) showed a total inhibition of the agonist induced amounts. Particularly in combination with isoproterenol low LDH activities reflected a high viability of hepatocytes. In conclusion, in primary hepatocyte cultures cAMP-mobilizing-agonists like isoproterenol and glucagon indicate an independent effect on the MT-metabolism. This is possibly due to the de novo synthesis of the protein because suppression by actinomycin D can be observed. However, cAMP-effectors do not seem to be involved in the induction of metallothionein because theophylline and dibutyryl-cAMP did not affect MT-metabolism by suppressing the phosphodiesterase or by stimulating the cAMP-cascade.

Fibroblasts isolated after fibrotic lung injury induce apoptosis of alveolar epithelial cells in vitro.

Primary lung fibroblasts were isolated from patients with idiopathic pulmonary fibrosis (HIPF), from normal human lung tissue (NH), from rats treated with 75% oxygen and paraquat (PA), and from normal adult rats (NR). Serum-free media conditioned by each fibroblast strain were tested on the human A549 cell line (HIPF and NH media) or on primary alveolar epithelial cells (AEC) isolated from normal adult rats (PA or NR media). Over 20-h incubation, HIPF- or PA-conditioned media induced DNA fragmentation and significant decreases in total recoverable DNA and cell number of A549 or AEC, respectively; NH or NR media had no significant effect relative to serum-free unconditioned media. Apoptosis of A549 and AEC was detected by altered nuclear morphology and was confirmed by terminal deoxynucleotidyl transferase-mediated bio-dUTP nick end labeling. The endonuclease inhibitors 10 microM aurintricarboxylic acid and 50 microM zinc inhibited HIPF-induced apoptosis of A549 cells by 68 and 71%, respectively. Both apoptosis and necrosis were induced by HIPF and PA media in a concentration-dependent manner. These results suggest that altered fibroblasts emerging during fibrotic lung injury release a soluble factor(s) capable of inducing cell death and net loss of AEC.

The effect of the acrodermatitis enteropathica mutation on zinc uptake in human fibroblasts.

The acrodermatitis enteropathica (AE) mutation affects intestinal zinc absorption. Our goal was to determine whether the AE mutation affects zinc uptake in human fibroblasts. Zinc uptake was determined during initial rates of uptake (10 min) following incubation in HEPES/saline buffer. Zinc uptake (from 0.25 to 1 microM) into normal fibroblasts was significantly greater than into the AE fibroblasts (p < 0.05). In order to identify factors that may alter cellular zinc uptake and be affected by the AE mutation, zinc uptake in the presence of albumin or bicarbonate was measured. Albumin restricted zinc uptake in both normal and AE fibroblasts, whereas bicarbonate stimulated zinc uptake in the normal fibroblasts. The effect of bicarbonate on zinc uptake in the AE fibroblasts was significantly reduced in both the Pronase-sensitive and Pronase-resistant compartments. Following loading of the fibroblasts with 1 microM zinc for 60 min, zinc efflux and retention were measured. The AE mutation did not affect zinc retention compared to normal fibroblasts. We conclude that the AE mutation affects both zinc binding to the cell surface and its translocation across the plasma membrane into the cell, possibly mediated through a defective anionic exchange mechanism.

Glycine-induced CA1 excitotoxicity in the rat hippocampal slice

We evaluated the effects of glycine exposure upon CA1 evoked response in the rat hippocampal slice. Exposure to 10 mM glycine for 16 min, produced rapid neuronal firing and increased orthodromic population spike (PS), followed by loss of CA1 neural transmission. Upon recovery, CA1 orthodromic and antidromic PS regained a mean of only 12 +/- 6% and 8 +/- 5% of initial amplitude. The electrophysiological pattern of glycine injury was similar to the excitotoxic damage produced by 8 min exposure to sodium glutamate 9 mM. L-Histidine, an inhibitor of glycine transport, exacerbated glycine-induced injury, just as dihydrokainic acid, a glutamate transport inhibitor, exacerbated glutamate-induced injury. The anticonvulsant felbamate (1.3 mM), as well as 100 microM zinc chloride, provided excellent protection from glycine-induced injury. 7-Chlorokynurenic acid appeared to be toxic. Blockers of the NMDA-associated ionic channel and methyl arginine, prevented loss of neural transmission, but did not prevent accompanying hyperexcitability. Only 10 mM magnesium sulfate provided full protection against 9 mM glutamate exposure. Perfusion with low calcium ACSF protected against both glycine and glutamate-induced injury. Thus, exposure to glycine resembled the excitotoxic effects of glutamate, but showed a different profile of protection. These results suggest that glycine elevations as occur under physiologic and pathologic conditions may modulate neuronal activity.

Zinc downmodulates thf GABAc receptor current in cone horizontal cells acutely isolated from the catfish retina.

1. We studied the effect of zinc on the gamma-aminobutyric acid-C (GABAC) receptor in acutely isolated catfish cone horizontal cells using the whole cell patch-clamp technique. 2. GABA activates the GABAC receptor with a half-activation concentration (EC50) of 2.99 microM. The Hill coefficient is 1.32. Desensitization of the receptor is evident when GABA concentration is > 3 microM. 3. Zinc downmodulates the GABAc receptor current, elicited by 30 microM GABA, with a half-inhibition concentration (IC50) of 8.20 microM. 4. The inhibition of zinc is both competitive and noncompetitive. In the presence of 10 microM zinc, the maximum GABA response was reduced to approximately 60 percent of control and the EC50 increased to 17.32 microM, whereas the Hill coefficient (1.39) was not significantly altered. 5. The steady-state block by zinc is virtually voltage independent. 6. These results suggest that the GABAC receptor of horizontal cells can be modulated by endogenous zinc found in photoreceptors.

Effect of nuclear Ca2+ uptake inhibitors on Ca(2+)-activated DNA fragmentation in rat liver nuclei.

The effect of nuclear Ca2+ uptake inhibitors on the Ca(2+)-activated DNA fragmentation in rat liver nuclei was investigated. The addition of Ca2+ (40 microM) into the reaction mixture containing liver nuclei in the presence of 2.0 mM ATP caused a remarkable increase in nuclear DNA fragmentation. This Ca(2+)-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 microM arachidonic acid, 2.0 mM NAD+, 10 microM zinc sulfate and 0.2 mM N-ethylmaleimide), which could inhibit Ca(2+)-ATPase activity and Ca2+ uptake in the nuclei, produced a significant inhibition of the Ca(2+)-activated DNA fragmentation in the nuclei. The results show that the Ca(2+)-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.

Zinc, vanadate and selenate inhibit the tri-iodothyronine-induced expression of fatty acid synthase and malic enzyme in chick-embryo hepatocytes in culture.

Insulin regulates the expression of genes involved in a variety of metabolic processes. In chick-embryo hepatocytes in culture, insulin amplifies the tri-iodothyronine (T3)-induced enzyme activity, and the level and rate of transcription of mRNA for both fatty acid synthase (FAS) and malic enzyme (ME). Insulin alone, however, has little or no effect on the expression of these genes. In chick-embryo hepatocytes, the mechanism by which insulin regulates the expression of these or other genes is not known. Several recent studies have compared the effects of zinc, vanadate and selenate on insulin-sensitive processes in an attempt to probe the mechanism of insulin action. Because zinc, vanadate and selenate mimic the effects of insulin on several processes, they have been termed insulin-mimetics. We have studied the effect of zinc, vanadate and selenate on the T3-induced expression of both FAS and ME. Like insulin, these agents had little or no effect on the basal activities for FAS and ME in chick-embryo hepatocytes in culture for 48 h. Unlike insulin, however, zinc, vanadate and selenate inhibited the T3-induced activities and mRNA levels of both FAS and ME. Maximal inhibition was achieved at concentrations of 50 microM zinc or vanadate, or 20 microM selenate. Zinc and vanadate also inhibited the T3-induced transcription of the FAS and ME genes. Although the mechanism of this inhibition is unknown, our results indicate that it is not mediated through inhibition of binding of T3 to its nuclear receptor nor through a general toxic effect. Thus zinc, vanadate and selenate are not insulin-mimetics under all conditions, and their effects on other insulin-sensitive processes may be fortuitous and unrelated to actions or components of the insulin signalling pathway.

The role of an inwardly rectifying chloride conductance in postsynaptic inhibition.

1. The relationship of the activation of a voltage-sensitive chloride conductance [GCl(V)] to the chloride transmembrane equilibrium potential (ECl) and the consequent role of this conductance in determining the effect of the gamma-aminobutyric acid-A (GABAA) receptor-mediated transmembrane chloride (Cl-) flux were investigated with the use of whole-cell recordings in the CA1 and dentate gyrus regions of adult rat hippocampal slice preparations. 2. GCl(V) was inwardly rectifying, with significant conductance only at membrane potentials more negative than ECl. For all tested neuronal Cl- concentrations, the activation of GCl(V) could be described by a Boltzman equation with an average half-activation voltage 15 mV negative to ECl, a slope factor of 14 mV, and a maximum conductance of 5 microS. There was no time-dependent inactivation of GCl(V). 3. GCl(V) was modulated by intracellular divalent cations. When magnesium was omitted from the electrode solution, the inward rectification of GCl(V) was unchanged, but the maximum amplitude of GCl(V) increased by a factor of 1.7. GCl(V) was blocked by bath application of 100 microM zinc (Zn2+), but not when 1-6 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) were present in the electrode solution. 4. GCl(V) was increased by 10 microM norepinephrine, and by activation of protein kinase A (PKA) with 1 mM 8-bromoadenosine cyclic monophosphate (8-Br cAMP). GCl(V) was blocked by activation of protein kinase C (PKC) with 10 microM phorbol 12,13-dibutyrate (PdBu) or 1-oleoyl-2-acetyl-sn-glycerol (OAG). 5. GCl(V) was present in all tested CA1 pyramidal neurons but no dentate gyrus neurons. In standard extracellular solution, the amplitude of GCl(V) was initially negligible but increased with recording time, suggesting that under normal conditions GCl(V) is blocked by an endogenous divalent cation or downregulated by PKC. 6. In current-clamp recordings, the steady-state resting membrane potential (RMP) diminished with Cl- loading, from -73 mV (4 mM electrode Cl-) to -27 mV (131 mM electrode Cl-). When GCl(V) was blocked with PdBu, there was no change in the RMP with Cl- loading. When electroneutral Cl- transport was blocked, voltage-clamp experiments using electrode Cl- concentrations of 4-131 mM demonstrated that ECl changed in parallel with the holding potential, but not when GCl(V) was blocked by PdBu.(ABSTRACT TRUNCATED AT 400 WORDS)

Zinc improves postischemic recovery of isolated rat hearts through inhibition of oxidative stress.

We studied the cardiac protective qualities of zinc in the postischemic isolated rat heart. Hearts, perfused with Krebs-Henseleit buffer with or without zinc-bis-histidinate, were subjected to 20 min of "no-flow" normothermic global ischemia. Pre- and postischemic treatment with 0, 10, 20, or 30 microM zinc resulted in concentration-dependent enhancement of postischemic function as evidenced by decreased end-diastolic pressure (37 +/- 3, 25 +/- 5, 17 +/- 5, and 8 +/- 2 mmHg, respectively) and increased recovery of developed systolic pressure (41 +/- 6, 59 +/- 17, 76 +/- 18, and 87 +/- 16 mmHg, respectively) and maximum rate of rise in pressure (+dP/dtmax; 823 +/- 141, 1,413 +/- 396, 1,700 +/- 450, and 2,157 +/- 407 mmHg/s, respectively) as well as decreased lactate dehydrogenase efflux from the hearts (peak: 1,002%, 840%, 580%, and 440%, respectively). Only preischemic treatment resulted in an intermediate protective effect, whereas treatment starting at reperfusion worsened postischemic damage. In hearts perfused with zinc throughout the experiment, prolongation of the preischemic treatment interval further enhanced postischemic recovery. With the use of salicylate as a trap for .OH, it was determined that zinc virtually eliminated the early postischemic "burst" of this species normally observed in this preparation. Atomic absorption studies demonstrated that hearts treated with 30 microM zinc contained 27% less copper than control hearts by the end of the reperfusion period. In control hearts, electron microscopy revealed swollen mitochondria with marked loss of inner matrix density, whereas morphology of postischemic zinc-treated hearts was essentially normal. These studies indicate that zinc possesses cardiac cytoprotective qualities and support the concept that this metal can decrease .OH formation by affecting copper reactivity.

Effect of pharmacologic doses of zinc on the therapeutic index of brain tumor chemotherapy with carmustine.

To evaluate the potential differential effect of pretreatment with pharmacologic doses of the trace element zinc on the chemosensitivity of glioma cells and bone marrow cells for carmustine (BCNU), we performed in vitro and in vivo studies of zinc toxicity as well as of the combined treatment with zinc and the anticancer drug. We studied the in vitro effects on established human and rat glioma cell lines using a microcolorimetric growth assay and on murine bone marrow using a clonogenic assay for committed progenitor cells of the granulocyte-monocyte lineage. Zinc exposures of up to 100 microM for 120 h did not influence the growth of six of seven human glioma cell lines. Only U87MG demonstrated statistically significant toxicity during high zinc exposure (100 microM over 120 h). Dose-response growth curves generated for BCNU did not show protection against the anticancer agents by a 48-h pretreatment with different zinc concentrations. The clonogenic capacity of bone marrow cells was slightly reduced by in vitro culture for 24 and 48 h. Although this effect appeared to be more prominent in the presence of zinc supplementation, overall a statistically significant inhibition was seen only after exposure to a concentration of 100 microM zinc over 48 h. As compared with chemotherapy alone, in vitro pretreatment with 50 microM zinc over 48 h followed by chemotherapy resulted in an increased number of colony-forming unit-granulocyte monocyte (CFU-GM): CFU-GM increased by a factor of 2 for BCNU (60 microM x 2 h). This statistically significant in vitro chemoprotection would translate into a dose-protection factor of 1.5, i.e., for the same level of myelosuppression, zinc pretreatment would allow administration of a 50% increased dose of BCNU. The in vivo studies were performed in an s.c. xenograft model of the human glioma cell line U87MG in athymic mice. The maximal tolerable pretreatment with zinc was determined to be a 10-day course of daily i.p. injections of 10 mg/kg ZnCl2. The subsequent i.p. administration of the dose lethal to 10% of the mice (LD10) and of a 1.5 x LD10 dose of BCNU resulted in less bone marrow toxicity in pretreated animals than in non-zinc-pretreated mice as determined in a CFU-GM assay. Glioma colony-forming efficiency (CFE) assays, on the other hand, did not show any zinc-related difference in the BCNU sensitivity of U87MG.(ABSTRACT TRUNCATED AT 400 WORDS)

Giant GABAB-mediated synaptic potentials induced by zinc in the rat hippocampus: paradoxical effects of zinc on the GABAB receptor.

The interaction of zinc with pre- and postsynaptic GABAB receptors was studied in adult rat hippocampal slices using intracellular recording in CA1 and CA3 pyramidal neurons. Zinc (50-300 microM) antagonized baclofen responses with a variable potency, whereas CGP-35348 (100 microM) or barium (300 microM) produced a more substantial and consistent inhibition. Zinc also induced giant GABAA-mediated depolarizing potentials (GDP) in these neurons. After blocking GABAA and excitatory synaptic transmission, monosynaptic hyperpolarizing inhibitory postsynaptic potentials (IPSP) mediated by GABAB receptors (IPSPB) were inhibited by CGP-35348 or barium; however, zinc increased the latency and prolonged the duration of the IPSPB and also induced the appearance of spontaneous giant GABAB-mediated hyperpolarizing potentials (GHP). In some cells, IPSPBs in zinc exhibited a multiphasic appearance. The early component was partially inhibited by 300 microM zinc and was followed by a late GHP. CGP-35348 at 100 microM inhibited the early monosynaptic IPSPB but not the GHP; however, at 300 microM both components were blocked. Paired-pulse inhibition of the IPSPB was used to assess the effect of zinc on presynaptic GABAB receptors. Neither the zinc-chelating agent CP94 (400 microM) nor zinc affected this phenomenon. CGP-35348, barium and polyvalent cations, such as cadmium, copper, cobalt, manganese, iron and aluminum, failed to induce giant potentials in hippocampal neurons. It is concluded that zinc is apparently unique in synchronizing the release of GABA to produce GDPs and GHPs.