Endogenous Metal Ions Research Articles

Binding of the Immunosuppressant Peptide Cyclosporine A to Calcium, Zinc and Copper. Is Cyclosporine A an Ionophore?

Cyclosporin A is an immunosuppressant drug that has made organ transplantation possible during the past decade. This conformationally flexible peptide, which interferes in an unknown way with calcium-dependent intracellular signalling events, has now been examined in aqueous and acetone solutions containing metal ions. By using electrospray mass spectrometry and nuclear magnetic resonance spectroscopy, cyclosporin A was found to bind directly to endogenous metal ions Ca2+, Zn2+ and Cu2+, with the highest affinity being for Ca2+. These observations raise the possibility that cyclosporins might directly regulate local concentrations of metal ions associated with intracellular signal transduction. This may be an important clue to the mechanism of immunoregulation by this and structurally related drugs.

Molecular and ionic mimicry of toxic metals.

The landmark review by Wetterhahn-Jennette (1) laid the foundation for this review. A recent review on mechanisms of metal transport (2) has also been an important literature resource. Wetterhahn-Jennette pointed out the structural similarities of many oxyanions and explained the toxicities of several metals in terms of oxyanions competing with endogenous oxyanions, phosphate, and sulfate in transport, enzymatic and receptor-mediated processes. She also introduced the term to describe the competition between many divalent endogenous and toxic metal ions in biological systems, such competition giving a mechanistic insight into the toxic action of certain metals. The term has been mainly confined to the field of immunology. In the context of this review, the term roolecular mimicry is restricted to those examples in which a toxic metal forms a complex with an endogenous ligand such that the resulting compound mimics the behavior of a normal substrate. An example to be discussed in some detail is the reaction of the methylmercury cation with the amino acid cysteine to form a compound that mimics the amino acid methionine and thus gains entry into the cell on an amino acid carrier. This review updates the literature on both oxyanion and divalent metal competition and introduces a new extension of molecular and ionic mimicry to complexes formed by mercury that are structurally similar to and biologi­ cally mimic endogenous molecules. This review is not intended to be an exhaustive account of all the literature but gives examples that illustrate the role of molecular and ionic mimicry in the action of the toxic metals.

Stability of gadolinium complexes in vitro and in vivo.

Gd(III) toxicity is such that, for in vivo use, the ion must be incorporated into stable, ionically bound complexes with organic chelating agents. Although metal-chelate-based contrast agents have been demonstrated safe in phase I-III studies, their potential toxicities must be considered, particularly in the development of new contrast media. The potential for adverse effects is thought to arise from in vivo dissociation into constituent metal ion and ligand. Release of Gd(III) from ligand may also be facilitated by endogenous metals [e.g., Zn(II)] that compete with Gd(III) for ligand-binding sites. In vivo complex stability toward dissociation depends on several closely balanced characteristics of the metal and ligand that maintain metal-chelate integrity during residence in the body. Among design strategies explored to minimize the potential for in vivo Gd(III) release are the use of ligands that: form Gd(III) complexes with high thermodynamic stabilities; have binding selectivity for Gd(III) over endogenous metal ions; incorporate rigid cyclic structures and form Gd(III) complexes with greater kinetic stabilities than their open-chain homologs; or incorporate slight excess ligand to "scavenge" loosely bound or free endogenous metal ions that might otherwise compete with Gd(III) for ligand binding.

Evaluation of the Ca2+ concentration in purified nerve terminals: relationship between Ca2+ homeostasis and synaptosomal preparation.

The presynaptic Ca2+ concentration ([Ca]i) was evaluated by studying intracellular free Ca2+ with quin-2 and fura-2 in synaptosomal preparations. The synaptosomal preparations were purified with hyperosmotic (sucrose) and isoosmotic (Percoll) density gradient centrifugation. Synaptosomes are most viable in the heavier fractions of the density gradients. These synaptosomal fractions exhibit the lowest [Ca]i, [204 +/- 2 nM for Percoll (C-band) synaptosomes, loaded at 30 degrees C with the acetoxymethyl ester of fura-2 (fura-2-AM)], a high stability during prolonged incubations at 37 degrees C, and a more potent response to membrane depolarization by elevated extracellular [K+]. [Ca]i measurement was critically dependent on dye loading, calibration, type of dye used, synaptosomal preparation, and incubation temperature (30 degrees or 37 degrees C). Loading quin-2 in synaptosomes inserts a considerable buffer component in the synaptosomal [Ca]i regulation, and consequently there is a quin-2 dependency of [Ca]i, independent of endogenous heavy metal ions. Use of fura-2 is preferable in synaptosomes, although above a critical fura-2-AM/protein ratio during loading ester hydrolysis is not complete, giving rise to errors in [Ca]i determination. Ionomycin is a selective tool to detect the presence of partially hydrolyzed esters and saturate indicators in the cytosol with Ca2+ for calibration. Parallel studies on lactate dehydrogenase and fura-2 fluorescence indicate that synaptosomal viability is very sensitive to prolonged incubations at 37 degrees C. This study shows the applicability of measuring steady-state [Ca]i and dynamic [Ca]i changes quantitatively in fura-2-loaded synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Metal-depleted Media on the Growth and Morphology of Saccharomyces rouxii and on the Status of Periplasmic Acid Phosphatase

Saccharomyces rouxii cells were maintained on yeast extract/neopeptone/glucose medium (YNG). Experimental medium was depleted of endogenous metal ions by repeated passage of YNG over a chelating ion-exchange resin. This treatment did not compromise the glucose or vitamin content but provided a basal medium to which essential salts could be added in defined concentrations. Accordingly it was possible to demonstrate growth requirements by this yeast species for K+, Mg2+, Zn2+ and Fe3+. Na+ would not replace K+. Additions of 1·0 μm-Ca2+, NH4+ and Na+, or 0·1 μm-Ni2+, Sn2+, Al3+, Co2+ or Cr3+ to a minimal, reconstituted medium were without effect. Cu2+ and Mn2+ additions were not required for growth but, like Mg2+, Zn2+ and Fe3+, they positively affected the biosynthesis of periplasmic acid phosphatase. Cells from iron-depleted medium had only 1 unit (g dry wt)−1 of native acid phosphatase activity although this could be stimulated 10-fold by addition of FeCl3 to washed cell suspensions before enzyme assay. Additions of 10–60 μm-FeCl3 to untreated YNG medium progressively increased the concentration of acid phosphatase after 3 d of culture to an upper value of 75 units (g dry wt)−1, and also increased the amount secreted into the growth medium. While unfortified YNG supported cells with only 28% of their acid phosphatase expressed (the bulk being subject to activation by Fe3+ in the assay medium) the cells from YNG fortified with 60 μm-FeCl3 displayed fully activated acid phosphatase. Cells from Mg2+-depleted medium exhibited bizarre, elongated cell shapes and those from Zn2+-deficient medium appeared somewhat angular by light microscopy. Fe3+-deficient cells had apparently normal morphology.

Opiate Receptor: Multiple Effects of Metal Ions

Abstract: The opiate antagonist [3H]diprenorphine ([3H]dip), a universal ligand at the μ, δ, and k opiate receptor subtypes, was used to study the effects of Ca‐II, Cu‐II, Mg‐II, Mn‐II, and Na+ on the rat cerebral opiate receptor. Two categories of effects were observed: (a) those on the binding rate constants and (b) those on binding capacity. (a) Sodium ions increased on‐ and off‐rates on [3H]dip with a rather small net change in receptor affinity. The effects of Na+ and the divalent ions Ca‐II, Mg‐II, and Mn‐II were antagonistic to each other. Ca‐II, Mg‐II, and the more effective Mn‐II decreased receptor association and dissociation rates, again with minimal changes in the overall binding affinity in washed membrane homogenates. Previous studies using equilibrium binding analysis alone failed to detect changes in [3H]dip binding kinetics caused by these metal ions. In untreated rat brain homogenates, however, Ca‐II (and to a lesser extent Mg‐II) decreased [3H]dip binding, an effect distinct from that on the binding rate constants in washed membrane homogenates. (b) In untreated, Tris‐buffer homogenates not containing external metal ions, a gradual decline in [3H]dip binding was observed. Cu‐II or an equivalent endogenous divalent metal ion was identified as a causative factor, and Mn‐II partially reversed this effect. Moreover, the addition of Mn‐II stabilized the [3H]dip binding sites at very low concentrations of the metal (nM to μM range) that did not change the binding rate constants and that were in the physiological range of Mn‐II in rat brain. This unique effect of Mn‐II may represent a physiological function in the regulation of the opiate receptor that is not shared by Mg‐II and Ca‐II. The opposite effects of Cu‐II and Mn‐II on the in vitro receptor stability may be related to their opposite pharmacological effect in vivo. Finally, multiple changes of the effects of the tested metal ions on [3H]dip binding were observed during in vitro membrane homogenate dilution, centrifugation, and washing. These changes indicate that the opiate receptor complex as it exists in vivo may lose some of its functions and control mechanisms in vitro.

Cation transport and specificity of ionomycin. Comparison with ionophore A23187 in rat liver mitochondria.

Based on the effects of ionomycin upon mitochondrial respiration, ionomycin was shown to be an effective ionophore for Ca2+ in rat liver mitochondria. The ionomycin-induced efflux of Ca2+ across the inner membrane was more sensitive to loading the mitochondria with Ca2+ than was efflux catalyzed by A23187. At saturating concentrations of Ca2+, the turnover number for ionomycin was 3- to 5-fold greater than that of A23187. Ionomycin catalyzed the efflux of mitochondrial Mg2+ at rates comparable to those observed with A23187. Ionomycin also mediated an efflux of K+ provided that the mitochondria were depleted of their endogenous divalent metal ions. The apparent turnover numbers for K+ efflux suggest that ionomycin is more specific for divalent metal ions than A23187.

Physiology and pharmacology of copper

Acid-sensing ion channels (ASICs) are known to distribute throughout the nervous system and serve important roles in various physiological and pathological processes. However, the properties of ASICs in the hypothalamus, an important region of diencephalon, are little known. We herein used whole-cell patch-clamp recordings to characterize proton-induced cation currents in cultured hypothalamic neurons of the rat, and attributed these transient inward currents to ASICs based on their electrophysiological and pharmacological properties. We further examined the effects of Cu2+, the third most abundant trace element, on ASICs in hypothalamic neurons. Our results showed that this divalent cation reversibly and concentration-dependently inhibited the amplitude of ASIC currents, and slowed down the desensitization of ASIC channels. Our results also displayed that Cu2+ modulated ASICs independent of change in membrane potential and extracellular protons, suggesting a noncompetitive mechanism. Furthermore, micromolar concentration of Cu2+ attenuated the acid-induced membrane depolarization. Taken together, our data demonstrate a modulatory effect of Cu2+ on ASICs in native hypothalamic neurons and suggest a role of this endogenous metal ion in negatively modulating the increased neuronal membrane excitability caused by activation of ASICs.