The effect of oxidized glutathione and its pharmacological analogue, glutoxim, on intracellular Ca2+ concentration in macrophages

The effect of oxidized glutathione (GSSG) and its pharmacological analogue, glutoxim, on intracellular Ca2+ concentration in rat peritoneal macrophages was investigated using Fura-2AM microfluorimetry. It was shown that both GSSG and glutoxim increased intracellular Ca2+ concentration inducing Ca(2+)-mobilization from thapsigargin-sensitive Ca(2+)-stores and subsequent Ca2+ entry into macrophages from external medium. Dithiothreitol, which reduces S-S-bonds in proteins, completely prevented or reversed the increase in the intracellular Ca2+ concentration induced by GSSG or glutoxim. It suggests that the increase in the intracellular Ca2+ concentration induced by GSSG or glutoxim can be mediated by their interactions with functionally important SH-groups of proteins involved in Ca(2+)-signaling. Two structurally different tyrosine kinase inhibitors, genistein and methyl-2,5-dihydroxycinnamate, prevented or completely reversed the increase in the intracellular Ca(2+)-concentration induced by GSSG or glutoxim. On the contrary, tyrosine phosphatase inhibitor, Na orthovanadate, enhanced the increase in the intracellular Ca2+ concentration evoked by oxidizing agents. The data suggest that tyrosine kinases and tyrosine phosphatases are involved in regulatory effects of GSSG and glutoxim on the intracellular Ca2+ concentration in macrophages.     

The effect of chlorpromazine on intracellular Ca<Superscript>2+</Superscript> concentration in macrophages

Using Fura-2AM microfluorimetry, it was shown for the first time that neuroleptic chlorpromazine causes intracellular Ca²⁺ concentration increase in macrophages due to Ca²⁺ mobilization from intracellular Ca²⁺ stores and subsequent Ca²⁺ entry from the external medium. Chlorpromazine-induced Ca²⁺ entry is inhibited by La³⁺ and 2-aminoethoxydiphenyl borate and is associated with Ca²⁺ store depletion.     

The involvement of actin cytoskeleton in glutoxim and molixan effect on intracellular Ca<Superscript>2+</Superscript>-concentration in macrophages

Glutoxim and molixan belong to new generation of disulfide-containing drugs with immunomodulatory, hepatoprotective and hemopoetic effect on cells. Using Fura-2AM microfluorimetry, two structurally distinct actin filament disrupters latrunculin B and cytochalasin D, and calyculin A, which causes actin filaments condensation under plasmalemma, we have shown the involvement of actin cytoskeleton in the intracellular Ca²⁺-concentration increase induced by glutoxim or molixan in rat peritoneal macrophages. Morphological data obtained with the use of rhodamine-phalloidine demonstrated that glutoxim and molixan cause the actin filaments reorganization in rat peritoneal macrophages.     

5-Lipoxygenase inhibitor zileuton inhibits Ca<Superscript>2+</Superscript>-responses induced by glutoxim and molixan in macrophages

Using Fura-2AM microfluorimetry, we have shown for the first time that 5-lipoxygenase specific inhibitor antiasthmatic agent zileuton significantly inhibits Ca²⁺-responses induced by glutoxim and molixan in macrophages. The results support 5-lipoxygenase involvement in the effect of glutoxim and molixan on intracellular Ca²⁺ concentration in macrophages and indicate the inadvisability of a combined use of drugs glutoxim and molixan and antiasthmatic agent zileuton.     

Effects of fluoride on the intracellular free Ca<Superscript>2+</Superscript> and Ca<Superscript>2+</Superscript>-ATPase of kidney

In the present study, the effect of fluoride on intracellular free calcium ([Ca2+]i) and Ca2+-ATPase of renal cells were examined. Some paradoxical experimental results about the mechanism of fluoride toxicity were observed. In vivo, 48 Wistar rats were divided into 4 groups, and half of rats were treated with sodium fluoride (NaF) by drinking water (per liter of tap water containing 100 mg F-). Compared with the respective control, the level of [Ca2+]i of the kidney in two fluoride-treated rats obviously increased (p < 0.05); and the activity of Ca2+-ATPase in 100 mg F-/L groups with a standard diet did not significantly increase, and the enzyme activity in 100-mg F-/L group with a low-calcium diet decreased significantly compared to the 100 mg F-/L group with a standard diet (p < 0.05). In vitro, renal tubular cells were cultured and respectively exposed to 1.0, 5.0, 7.5, and 12.5 mg/L fluoride in the culture medium. Results showed the significantly elevated activity of Ca2+-ATPase in the cells exposed to 1.0 and 5.0 mg/L fluoride (p < 0.05), and this enzyme activity indicated inhibitory trend in cells of the 7.5- and 12.5-mg/L fluoride-treated group. To sum up, the effect of fluoride on Ca2+-ATPase is a similar to a dose-effect relationship phenomenon characterized by low-dose stimulation and high-dose inhibition, and the increase of [Ca2+]i probably plays a key role on the mechanism of renal injury in fluorosis.     

Sigma-1 receptor antagonist haloperidol attenuates Ca<Superscript>2+</Superscript> responses induced by glutoxim and molixan in macrophages

Using Fura-2AM microfluorimetry, we have shown for the first time that sigma-1 receptor antagonist, antipsychotic haloperidol, significantly inhibits glutoxim- and molixan-induced Ca²⁺-response in peritoneal macrophages. These results indicate possible involvement of sigma-1 receptors in the signal cascade induced by glutoxim or molixan and leading to intracellular Ca²⁺ concentration increase in macrophages.     

Anoxia-induced elevation of cytosolic Ca<Superscript>2+</Superscript> concentration depends on different Ca<Superscript>2+</Superscript> sources in rice and wheat protoplasts

The anoxia-dependent elevation of cytosolic Ca²⁺ concentration, [Ca²⁺]_cyt, was investigated in plants differing in tolerance to hypoxia. The [Ca²⁺]_cyt was measured by fluorescence microscopy in single protoplasts loaded with the calcium-fluoroprobe Fura 2-AM. Imposition of anoxia led to a fast (within 3 min) significant elevation of [Ca²⁺]_cyt in rice leaf protoplasts. A tenfold drop in the external Ca²⁺ concentration (to 0.1 mM) resulted in considerable decrease of the [Ca²⁺]_cyt shift. Rice root protoplasts reacted upon anoxia with higher amplitude. Addition of plasma membrane (verapamil, La³⁺ and EGTA) and intracellular membrane Ca²⁺-channel antagonists (Li⁺, ruthenium red and cyclosporine A) reduced the anoxic Ca²⁺-accumulation in rice. Wheat protoplasts responded to anoxia by smaller changes of [Ca²⁺]_cyt. In wheat leaf protoplasts, the amplitude of the Ca²⁺-shift little depended on the external level of Ca²⁺. Wheat root protoplasts were characterized by a small shift of [Ca²⁺]_cyt under anoxia. Plasmalemma Ca²⁺-channel blockers had little effect on the elevation of cytosolic Ca²⁺ in wheat protoplasts. Intact rice seedlings absorbed Ca²⁺ from the external medium under anoxic treatment. On the contrary, wheat seedlings were characterized by leakage of Ca²⁺. Verapamil abolished the Ca²⁺ influx in rice roots and Ca²⁺ efflux from wheat roots. Anoxia-induced [Ca²⁺]_cyt elevation was high particularly in rice, a hypoxia-tolerant species. In conclusion, both external and internal Ca²⁺ stores are important for anoxic [Ca²⁺]_cyt elevation in rice, whereas the hypoxia-intolerant wheat does not require external sources for [Ca²⁺]_cyt rise. Leaf and root protoplasts similarly responded to anoxia, independent of their organ origin.     

Molecular evolution of intracellular Ca<Superscript>2+</Superscript>-dependent proteases

The structural features and evolutionary interrelationships of the intracellular Ca²⁺-dependent cysteine enzymes calpains, proteases of the family C2 (EC 3.4.22.17), are considered. A variety of identified sequences of calpains and calpain-like polypeptides found in organisms of different taxons, from the protozoa to mammals, are described. Calpains of the major evolutionary groups, typical and atypical, are classified by the analysis of their phylogenetic tree and are differentiated due to the presence of the calmodulin-like Ca²⁺-binding domain. It is shown that, along with enzymes having “advanced” characteristics (heterodimeric structure, presence of tissue-specific isoforms and splice variants, regulation by the endogenous inhibitor calpastatin, and others), higher organisms contain homologues of calpains of lower eukaryotes. A high degree of homology of the catalytic domain of calpains and the variable structure of other functional domains indicate that calpains are implicated in various physiological processes with the retention of their regulatory role.     

The effects of Weichangshu tablet on intracellular Ca<Superscript>2+</Superscript> concentration in cultured rat gastrointestinal smooth muscle cells

The objective of this study was to explore the effects of Weichangshu tablets on free Ca²⁺ concentration of the gastrointestinal smooth muscle cells of rats and the molecular mechanism of the Weichangshu tablets. Cultured SD rat gastrointestinal smooth muscle cells were divided into control group, Cisapride group, Weichangshu group, and control + ethylene glycol tetraacetic acid (EGTA) group, Cisapride + EGTA group, and Weichangshu + EGTA group. Laser scanning microscope and spectrophotometer detection were applied to detect the calcium concentration. The calcium concentrations in Weichangshu group and Cisapride group significantly increased vs. control, and those in Weichangshu group were higher than those in Cisapride group. Ca²⁺ concentration in Weichangshu + EGTA group, Cisapride + EGTA group vs. control + EGTA group were not significantly different. Weichangshu could increase gastrointestinal smooth muscle free Ca²⁺ concentration, and this role may be achieved through the promotion of cells within the flow of calcium ions.     

The effects of Tl<Superscript>+</Superscript> ions on the dynamics of intracellular Ca<Superscript>2+</Superscript> in rat cardiomyocytes

The effects of Т1⁺ ions on the dynamics of intracellular Cа²⁺ in neonatal rat cardiomyocytes have been studied. It was shown for the first time that application of Т1⁺ led to an uncontrolled increase in [Cа²⁺]_i in cells. Moreover, the ability of Т1⁺ to increase [Cа²⁺]_i depended on the Т1⁺ concentration used and the time of exposure to the cells. The increase in [Cа²⁺]_i was related to the entry of Cа²⁺ from the extracellular medium. Thallium did not release Cа²⁺ from intracellular stores. The thallium-induced increase in [Cа²⁺]_i was not inhibited by nifedipine. It is possible that L-channels do not participate in the processes of thalliuminduced increase in [Cа²⁺]_i. It is assumed that the thallium ions-induced calcium overload in cardiomyocytes may contribute to the toxic effect of Т1⁺ on the myocardium.     

Ca<Superscript>2+</Superscript> binding protein-1 inhibits Ca<Superscript>2+</Superscript> currents and exocytosis in bovine chromaffin cells

Summary Calcium binding protein-1 (CaBP1) is a calmodulin like protein shown to modulate Ca²⁺ channel activities. Here, we explored the functions of long and short spliced CaBP1 variants (L- and S-CaBP1) in modulating stimulus-secretion coupling in primary cultured bovine chromaffin cells. L- and S-CaBP1 were cloned from rat brain and fused with yellow fluorescent protein at the C-terminal. When expressed in chromaffin cells, wild-type L- and S-CaBP1s could be found in the cytosol, plasma membrane and a perinuclear region; in contrast, the myristoylation-deficient mutants were not found in the membrane. More than 20 and 70% of Na⁺ and Ca²⁺ currents, respectively, were inhibited by wild-type isoforms but not myristoylation-deficient mutants. The [Ca²⁺] _i response evoked by high K⁺ buffer and the exocytosis elicited by membrane depolarizations were inhibited only by wild-type isoforms. Neuronal Ca²⁺ sensor-1 and CaBP5, both are calmodulin-like proteins, did not affect Na⁺, Ca²⁺ currents, and exocytosis. When expressed in cultured cortical neurons, the [Ca²⁺] _i responses elicited by high-K⁺ depolarization were inhibited by CaBP1 isoforms. In HEK293T cells cotransfected with N-type Ca²⁺ channel and L-CaBP1, the current was reduced and activation curve was shifted positively. These results demonstrate the importance of CaBP1s in modulating the stimulus-secretion coupling in excitable cells. M.-L. Chen and Y.-C. Chen contributed equally to this study     

L-type Ca<Superscript>2+</Superscript> channel and Ca<Superscript>2+</Superscript>-permeable nonselective cation channel as a Ca<Superscript>2+</Superscript> conducting pathway in myocytes isolated from the cricket lateral oviduct

The Ca²⁺-conducting pathway of myocytes isolated from the cricket lateral oviduct was investigated by means of the whole-cell patch clamp technique. In voltage-clamp configuration, two types of whole cell inward currents were identified. One was voltage-dependent, initially activated at –40 mV and reaching a maximum at 10 mV with the use of 140 mM Cs²⁺-aspartate in the patch pipette and normal saline in the bath solution. Replacement of the external Ca²⁺ with Ba²⁺ slowed the current decay. Increasing the external Ca²⁺ or Ba²⁺ concentration increased the amplitude of the inward current and the current–voltage (I–V) relationship was shifted as expected from a screening effect on negative surface charges. The inward current could be carried by Na⁺ in the absence of extracellular Ca²⁺. Current carried by Na⁺ (I _Na) was almost completely blocked by the dihydropyridine Ca²⁺ channel antagonist, nifedipine, suggesting that the I _Na is through voltage-dependent L-type Ca²⁺ channels. The other inward current is voltage-independent and its I–V relationship was linear between –100 mV to 0 mV with a slight inward rectification at more hyperpolarizing membrane potentials when 140 mM Cs⁺-aspartate and 140 mM Na⁺-gluconate were used in the patch pipette and in the bath solution, respectively. A similar current was observed even when the external Na⁺ was replaced with an equimolar amount of K⁺ or Cs⁺, or 50 mM Ca²⁺ or Ba²⁺. When the osmolarity of the bath solution was reduced by removing mannitol from the bath solution, the inward current became larger at negative potentials. The I–V relationship for the current evoked by the hypotonic solution also showed a linear relationship between –100 mV to 0 mV. Bath application of Gd³⁺ (10 M) decreased the inward current activated by membrane hyperpolarization. These results clearly indicate that the majority of current activated by a membrane hyperpolarization is through a stretch-activated Ca²⁺-permeable nonselective cation channel (NSCC). Here, for the first time, we have identified voltage-dependent L-type Ca²⁺ channel and stretch-activated Ca²⁺-permeable NSCCs from enzymatically isolated muscle cells of the cricket using the whole-cell patch clamp recording technique.Abbreviations I _Ca Ca²⁺ current - I _Na Na⁺ current - I–V current–voltage - NSCC nonselective cation channel Communicated by G. Heldmaier     

Influence of Ca<Superscript>2+</Superscript> Oscillatory Influx on Membrane Ca<Superscript>2+</Superscript>-ATPase Activity: a Kinetic Model

A kinetic model for the membrane Ca²⁺-ATPase is considered. The catalytic cycle in the model is extended by enzyme auto-inhibition and by oscillatory calcium influx. It is shown that the conductive enzyme activity can be registered as damped or sustained Ca²⁺ pulses similar to observed experimentally. It is shown that frequency variations in Ca²⁺ oscillatory influx induce changes of pulsating enzyme activity. Encoding is observed for the signal frequency into a number of fixed levels of sustained pulses in the enzyme activity. At certain calcium signal frequencies, the calculated Ca²⁺-ATPase conductivity demonstrates chaotic multi-level pulses, similar to those observed experimentally.__________Translated from Biokhimiya, Vol. 70, No. 4, 2005, pp. 539–544.Original Russian Text Copyright © 2005 by Goldstein, Mayevsky, Zakrjevskaya.     

Thiamine Deficiency Increases Ca<Superscript>2+</Superscript> Current and Ca<Subscript>V</Subscript>1.2 L-type Ca<Superscript>2+</Superscript> Channel Levels in Cerebellum Granular Neurons

Thiamine (vitamin B1) is co-factor for three pivotal enzymes for glycolytic metabolism: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase. Thiamine deficiency leads to neurodegeneration of several brain regions, especially the cerebellum. In addition, several neurodegenerative diseases are associated with impairments of glycolytic metabolism, including Alzheimer’s disease. Therefore, understanding the link between dysfunction of the glycolytic pathway and neuronal death will be an important step to comprehend the mechanism and progression of neuronal degeneration as well as the development of new treatment for neurodegenerative states. Here, using an in vitro model to study the effects of thiamine deficiency on cerebellum granule neurons, we show an increase in Ca²⁺ current density and Ca_V1.2 expression. These results indicate a link between alterations in glycolytic metabolism and changes to Ca²⁺ dynamics, two factors that have been implicated in neurodegeneration.