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.     

Amitriptyline 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 agonist, tricyclic antidepressant amitriptyline, significantly inhibits glutoxim- and molixan-induced Ca²⁺-responses in rat peritoneal macrophages. The results suggest possible involvement of sigma-1 receptors in the signaling cascade induced by glutoxim or molixan and leading to intracellular Ca²⁺ concentration increase in 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.     

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.     

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.     

Inhibitors of the Metabolism of Arachidonic Acid Suppress Ca<Superscript>2+</Superscript> Responses Induced by Trifluoperazine in Macrophages

The influence of the neuroleptic trifluoperazine on the intracellular concentration of Ca²⁺ in macrophages of rats was studied using a Fura-2AM fluorescent Ca²⁺ probe. It was found that trifluoperazine causes a dose-dependent increase in the intracellular Ca²⁺ concentration associated with Ca²⁺ mobilization from intracellular Ca²⁺ stores and subsequent entry of Ca²⁺ into peritoneal macrophages of rats. It was also shown that inhibitors of phospholipase A₂ (4-bromophenacyl bromide, prednisolone, and dexamethasone), cyclooxygenases (aspirin and indomethacin), and lipoxygenases (caffeic acid, zileuton, and baicalein) suppress Ca²⁺ responses induced by trifluoperazine in macrophages. The data obtained indicate the participation of enzymes and/or products of the cascade of arachidonic acid metabolism in the influence of trifluoperazine on the intracellular concentration of Ca²⁺ in peritoneal 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.     

Ca<Superscript>2+</Superscript> channels and Ca<Superscript>2+</Superscript> signals involved in abiotic stress responses in plant cells: recent advances

Calcium (Ca²⁺) signals are essential transducers and regulators in many adaptive and developmental processes in plants. Protective responses of plants to a variety of environmental stress factors are mediated by transient changes of Ca²⁺ concentration in plant cells. Ca²⁺ ions are quickly transported by channel proteins present on the plasma membrane. During responses to external stimuli, various signal molecules are transported directly from extracellular to intracellular compartments via Ca²⁺ channel proteins. Three types of Ca²⁺ channels have been identified in plant cell membranes: voltage-dependent Ca²⁺-permeable channels (VDCCs), which is sorted to depolarization-activated Ca²⁺-permeable channels (DACCs) and hyperpolarization-activated Ca²⁺-permeable channels (HACCs), voltage-independent Ca²⁺-permeable channels (VICCs). They make functions in the abiotic stress such as TPCs, CNGCs, MS channels, annexins which distribute in the organelles, plasma membrane, mitochondria, cytosol, intracelluar membrane. This review summarizes recent advances in our knowledge of many types of Ca²⁺ channels and Ca²⁺ signals involved in abiotic stress resistance and responses in plant cells.     

Phospholipase A<Subscript>2</Subscript> Inhibitors Modulate the Effect of Trifluoperazine on the Intracellular Ca<Superscript>2+</Superscript> Concentration in Macrophages

Using Fura-2AM microfluorimetry, it was shown for the first time that phospholipase A₂ inhibitors 4-bromophenacyl bromide and glucocorticosteroids prednisolone and dexamethasone attenuate Ca²⁺ responses induced by neuroleptic trifluoperazine in macrophages. The results suggest the involvement of phospholipase A₂ and arachidonic acid metabolism cascade in the effect of trifluoperazine on intracellular Ca²⁺ concentration in macrophages.     

Trifluoperazine Attenuates Store-Dependent Ca<Superscript>2+</Superscript> Entry in Macrophages

Using Fura-2AM microfluorimetry, we have shown for the first time that preincubation of macrophages with the calsequestrin inhibitor neuroleptic trifluoperazine leads to a significant inhibition of the store-dependent Ca²⁺ entry induced by endoplasmic Ca²⁺-ATPase inhibitors thapsigargin or cyclopiazonic acid in rat peritoneal macrophages. The results suggest calsequestrin involvement in the regulation of the store-dependent Ca²⁺ entry in macrophages.     

Intracellular Ca<Superscript>2+</Superscript> Pools and Fluxes in Cardiac Muscle-Derived H9c2 Cells

Relevant Ca²⁺ pools and fluxes in H9c2 cells have been studied using fluorescent indicators and Ca²⁺-mobilizing agents. Vasopressin produced a cytoplasmic Ca²⁺ peak with half-maximal effective concentration of 6 nM, whereas thapsigargin-induced Ca²⁺ increase showed half-maximal effect at 3 nM. Depolarization of the mitochondrial inner membrane by protonophore was also associated with an increase in cytoplasmic Ca²⁺. Ionomycin induced a small and sustained depolarization, while thapsigargin had a small but transient effect. The thapsigargin-sensitive Ca²⁺ pool was also sensitive to ionomycin, whereas the protonophore-sensitive Ca²⁺ pool was not. The vasopressin-induced cytoplasmic Ca²⁺ signal, which caused a reversible discharge of the sarco-endoplasmic reticulum Ca²⁺ pool, was sensed as a mitochondrial Ca²⁺ peak but was unaffected by the permeability transition pore inhibitor cyclosporin A. The mitochondrial Ca²⁺ peak was affected by cyclosporin A when the Ca²⁺ signal was induced by irreversible discharge of the intracellular Ca²⁺ pool, i.e., adding thapsigargin. These observations indicate that the mitochondria interpret the cytoplasmic Ca²⁺ signals generated in the reticular store.     

The Role of the Golgi-Resident SPCA Ca<Superscript>2+</Superscript>/Mn<Superscript>2+</Superscript> Pump in Ionic Homeostasis and Neural Function

Recent evidence highlights the functional importance of the Golgi apparatus (GA) in neurological diseases. The functions of the mammalian GA, in addition to the processing and transport of cargo, also include ionic homeostasis. Besides Ca²⁺-release channels which serves GA as an agonist-sensitive intracellular Ca²⁺ store, and Ca²⁺-binding proteins, the GA contains Ca²⁺-uptake mechanisms consisting of the well-known sarco-endoplasmic reticulum Ca²⁺-transport ATPases and the much less characterized secretory-pathway Ca²⁺-transport ATPases (SPCA). SPCA can transport both Ca²⁺ and Mn²⁺ into the Golgi lumen and therefore is involved in the cytosolic and intra-Golgi Ca²⁺ and Mn²⁺ homeostasis. It has shown that both of the mRNA and protein of SPCAs are highly expressed in brain. In addition, brain is the region with the highest activity of SPCA isoforms, which may be related to the involvement of Ca²⁺ and Mn²⁺ homeostasis in neural functions. In this review, we compile some recent findings showing that the SPCA isoform plays a much more important role in intracellular ionic homeostasis than previously anticipated and illustrating the involvement of SPCA isoforms in certain neurophysiological or neuropathological process. We are interested in gaining insight into the intricate role of the SPCA pumps to explain the GA-specific functions in neurological disorders.     

Sigma-1 Receptor Antagonist Haloperidol Attenuates Store-Dependent Ca<Superscript>2+</Superscript> Entry in Macrophages

Using Fura-2AM microfluorimetry, we have shown for the first time that preincubation of macrophages with sigma-1 receptor antagonist haloperidol leads to a significant inhibition of the store-dependent Ca²⁺ entry induced by endoplasmic Ca²⁺-ATPase inhibitors thapsigargin or cyclopiazonic acid in rat peritoneal macrophages. The results suggest the involvement of the sigma-1 receptor in the regulation of storedependent Ca²⁺ entry in macrophages.     

Nicotine-induced Ca<Superscript>2+</Superscript>-myristoyl Switch of Neuronal Ca<Superscript>2+</Superscript> Sensor VILIP-1 in Hippocampal Neurons: A Possible Crosstalk Mechanism for Nicotinic Receptors

Visinin-like protein (VILIP-1) belongs to the neuronal Ca²⁺ sensor family of EF-hand Ca²⁺-binding proteins that regulate a variety of Ca²⁺-dependent signal transduction processes in neurons. It is an interaction partner of α4β2 nicotinic acetylcholine receptor (nAChR) and increases surface expression level and agonist sensitivity of the receptor in oocytes. Nicotine stimulation of nicotinic receptors has been reported to lead to an increase in intracellular Ca²⁺ concentration by Ca²⁺-permeable nAChRs, which in turn might lead to activation of VILIP-1, by a mechanism described as the Ca²⁺-myristoyl switch. It has been postulated that this will lead to co-localization of the proteins at cell membranes, where VILIP-1 can influence functional activity of α4-containing nAChRs. In order to test this hypothesis we have investigated whether a nicotine-induced and reversible Ca²⁺-myristoyl switch of VILIP-1 exists in primary hippocampal neurons and whether pharmacological agents, such as antagonist specific for distinct nAChRs, can interfere with the Ca²⁺-dependent membrane localization of VILIP-1. Here we report, that only α7- but not α4-containing nAChRs are able to elicit a Ca²⁺-dependent and reversible membrane-translocation of VILIP-1 in interneurons as revealed by employing the specific receptor antagonists dihydro-beta-erythroidine and methylallylaconitine. The nAChRs are associated with processes of synaptic plasticity in hippocampal neurons and they have been implicated in the pathology of CNS disorders, including Alzheimer’s disease and schizophrenia. VILIP-1 might provide a novel functional crosstalk between α4- and α7-containing nAChRs.     

cAMP controls cytosolic Ca<Superscript>2+</Superscript> levels in <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis>

Background  

Differentiating Dictyostelium discoideum amoebae respond upon cAMP-stimulation with an increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) that is composed of liberation of stored Ca²⁺ and extracellular Ca²⁺-influx. In this study we investigated whether intracellular cAMP is involved in the control of [Ca²⁺]_i.     

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.     

Store-Dependent Entry of Ca<Superscript>2+</Superscript> into Sensory Neurons of the Rat

We studied store-dependent (activated by depletion of the endoplasmic reticulum, ER, store) entry of Ca²⁺ from the extracellular medium into neurons of the rat spinal ganglia (small- and medium-sized cells; diameter, 18 to 36 μm). Activation of ryanodine-sensitive receptors of the ER in the studied neurons superfused by Tyrode solutions containing Ca²⁺ or with no Ca²⁺ was provided by application of 10 mM caffeine. The decay phase of caffeine-induced calcium transients in a Ca²⁺-containing solution was significantly longer than that in a Ca²⁺-free solution. This fact allows us to suppose that such a phenomenon is determined by Ca²⁺ entry into the neuron from the extracellular medium activated by caffeine-induced depletion of the ER store. Substitution of Ca²⁺-free extracellular solution by Ca²⁺-containing Tyrode solution, after depletion of the ER stores induced by applications of 100 nM ryanodine, 200 μM ATP, or 1 μM thapsigargin, resulted in increases in the concentration of intracellular Ca²⁺. These observations allow us to postulate that store-dependent Ca²⁺ entry into the studied neurons is activated after depletion not only of the inositol trisphosphate-sensitive ER store but also of the ryanodine-sensitive store. This entry also occurs after blocking of ATPases of the ER by thapsigargin. The kinetic characteristics of the rising phase of store-dependent Ca²⁺ entry induced by depletion of the ER stores under the influence of various agents are dissimilar; this can be related to different mechanisms of activation of such signals and/or to a compartmental organization of the ER. Neirofiziologiya/Neurophysiology, Vol. 37, No. 3, pp. 277–283, May–June, 2005.     

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     

Boric acid inhibits stored Ca<Superscript>2+</Superscript> release in DU-145 prostate cancer cells

Boron (B) is a developmental and reproductive toxin. It is also essential for some organisms. Plants use uptake and efflux transport proteins to maintain homeostasis, and in humans, boron has been reported to reduce prostate cancer. Ca²⁺ signaling is one of the primary mechanisms used by cells to respond to their environment. In this paper, we report that boric acid (BA) inhibits NAD⁺ and NADP⁺ as well as mechanically induced release of stored Ca²⁺ in growing DU-145 prostate cancer cells. Cell proliferation was inhibited by 30% at 100μM, 60% at 250μM, and 97% at 1,000μM BA. NAD⁺-induced Ca²⁺ transients were partly inhibited at 250μM BA and completely at 1,000μM BA, whereas both NADP⁺ and mechanically induced transients were inhibited by 1,000μM BA. Expression of CD38 protein increased in proportion to BA exposure (0–1,000μM). In vitro mass spectrometry analysis showed that BA formed adducts with the CD38 products and Ca²⁺ channel agonists cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). Vesicles positive for the Ca²⁺ fluorophore fluo-3 acetoxymethyl ester accumulated in cells exposed to 250 and 1,000μM BA. The BA analog, methylboronic acid (MBA; 250 and 1,000μM), did not inhibit cell proliferation or NAD⁺, NADP⁺, or mechanically stimulated Ca²⁺ store release. Nor did MBA increase CD38 expression or cause the formation of intracellular vesicles. Thus, mammalian cells can distinguish between BA and its synthetic analog MBA and exhibit graded concentration-dependent responses. Based on these observations, we hypothesize that toxicity of BA stems from the ability of high concentrations to impair Ca²⁺ signaling.