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.     

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.     

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.     

Methyl-β-cyclodextrin modulates thapsigargin-induced 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 methyl-β-cyclodextrin, inducing cholesterol extraction from membranes and raft disruption, leads to significant inhibition of thapsigargin-induced store-dependent Ca²⁺ entry in rat peritoneal macrophages. In contrast, macrophage treatment with methyl-β-cyclodextrin after Ca²⁺ entry mechanisms were activated by store depletion by thapsigargin application leads to potentiation of subsequent store-dependent Ca²⁺ entry. The results suggest that intact lipid rafts are necessary for the activation but not the maintenance of store-dependent Ca²⁺ entry 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.     

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 oxidized glutathione and its pharmacological analogue glutoxim on intracellular Ca<Superscript>2+</Superscript> concentration in macrophages Ca<Superscript>2+</Superscript>

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

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.     

The mechanism of neuroprotection by positive modulation of Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channels of cerebellar neurons in primary culture

Here we show that positive modulators (CyPPA and NS309) of Ca²⁺-activated K⁺ channels of small (SK) and intermediate (IK) conductances in cerebellar neurons decrease glutamate-evoked Ca²⁺ entry into neurons independently on the presence of Mg²⁺ in extracellular media. An analysis of neuronal viability after long-term (240 min) glutamate treatments demonstrated neuroprotective action of CyPPA and NS309. Extracellular Mg²⁺ did not protect neurons from apoptosis during prolonged treatment with glutamate. Activation of SK and IK channels results in local membrane hyperpolarization, which enhances Mg²⁺ block of NMDA receptors and reduces activation of voltage-dependent Ca²⁺ channels, which can explain neuroprotection caused by CyPPA or NS309. The obtained results reveal an important role Ca²⁺-activated K⁺ channels of small and intermediate conductance in the regulation of Ca²⁺ entry into cerebellar neurons via NMDA receptors and voltage-gated Ca²⁺ channels.     

Features of Ca<Superscript>2+</Superscript> signaling in proliferating and in differentiating murine myoblasts

The state of the Ca²⁺ signaling system has been assessed in proliferating and in differentiating C2C12 myoblasts. Proliferating myoblasts exhibit no features of a functional system: the intracellular ATP-controlled Ca²⁺ store is low (perhaps only mitochondrial) and no Ca²⁺ entry from the medium is registered upon its exhaustion, there is no cytosolic response to KCl-induced depolarization. The Ca²⁺ signaling system starts to form at the early stages of differentiation (within 10 h after transfer of cells to the differentiation medium). This is seen as appearance of capacitive and voltage-dependent Ca²⁺ entry and its accumulation in the endoplasmic reticulum. A small contribution to the ATP-evoked rise in cytosolic Ca²⁺ is also made by mitochondria.     

Involvement of Ca<Superscript>2+</Superscript> signaling in tachykinin-mediated contractile responses in swine trachea

Summary Neuropeptide tachykinins, present within sensory nerves, have been implicated as neurotransmitters involved in nonadrenergic and noncholinergic airway muscle contraction. The signal transduction pathways of tachykinins on muscle contraction and Ca²⁺ mobilization were investigated in swine trachea. Tachykinins, substance P (SP) and neurokinin A (NKA), concentration (1 nM to 1 μM)-dependently induced contractile responses with removal of epithelium, whereas neurokinin B (NKB) did not alter the muscle tension. The SP- and NKA-evoked muscle contractions were inhibited by NK1-R antagonist L732138, but not by either NK2-R antagonist MDL29913 or NK3-R antagonist SB218795. Consistently, SP-elicited increase in [Ca²⁺]_i was abolished by NK1-R antagonist, neither by NK2-R nor NK3-R antagonists. The SP-induced muscular responses were significantly inhibited by L-type Ca²⁺ channel blocker verapamil and withdrawal of external Ca²⁺. Caffeine (10 mM) or ryanodine (50 μM) also partly suppressed the SP-induced muscle responses. Inhibition of inositol 1,4,5-trisphosphate (InsP₃) receptor with 2-APB (75 μM) potently attenuated SP-evoked Ca²⁺ mobilization and muscle contraction, which was further inhibited by 2-APB under Ca²⁺-free external solution, but not completely. Unexpectedly, simultaneous blockade of InsP₃ receptor and ryanodine receptor (RyR) by 2-APB and ryanodine enhanced SP-evoked muscle contraction and Ca²⁺ mobilization. This potentiation was virtually abolished by removal of external Ca²⁺, suggesting native Ca²⁺ channels may contribute to this phenomenon. These results demonstrate that tachykinins produce a potent muscle contraction associated with Ca²⁺ mobilization via tachykinin NK1- R-dependent activation of multiple signal transduction pathways involving Ca²⁺ influx and release of Ca²⁺ from InsP₃- and ryanodine-sensitive Ca²⁺ stores. Blockade of both InsP₃ receptor and RyR enhances the Ca²⁺ influx through native Ca²⁺ channels in plasma membrane, which is crucial to Ca²⁺ signaling in response to NK1 receptor activation.     

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.     

Nanosecond Electric Pulses: A Novel Stimulus for Triggering Ca<Superscript>2+</Superscript> Influx into Chromaffin Cells Via Voltage-Gated Ca<Superscript>2+</Superscript> Channels

Exposing bovine chromaffin cells to a single 5 ns, high-voltage (5 MV/m) electric pulse stimulates Ca²⁺ entry into the cells via L-type voltage-gated Ca²⁺ channels (VGCC), resulting in the release of catecholamine. In this study, fluorescence imaging was used to monitor nanosecond pulse-induced effects on intracellular Ca²⁺ level ([Ca²⁺]_i) to investigate the contribution of other types of VGCCs expressed in these cells in mediating Ca²⁺ entry. ω-Conotoxin GVIA and ω-agatoxin IVA, antagonists of N-type and P/Q-type VGCCs, respectively, reduced the magnitude of the rise in [Ca²⁺]_i elicited by a 5 ns pulse. ω-conotoxin MVIIC, which blocks N- and P/Q-type VGCCs, had a similar effect. Blocking L-, N-, and P\Q-type channels simultaneously with a cocktail of VGCC inhibitors abolished the pulse-induced [Ca²⁺]_i response of the cells, suggesting Ca²⁺ influx occurs only via VGCCs. Lowering extracellular K⁺ concentration from 5 to 2 mM or pulsing cells in Na⁺-free medium suppressed the pulse-induced rise in [Ca²⁺]_i in the majority of cells. Thus, both membrane potential and Na⁺ entry appear to play a role in the mechanism by which nanoelectropulses evoke Ca²⁺ influx. However, activation of voltage-gated Na⁺ channels (VGSC) is not involved since tetrodotoxin (TTX) failed to block the pulse-induced rise in [Ca²⁺]_i. These findings demonstrate that a single electric pulse of only 5 ns duration serves as a novel stimulus to open multiple types of VGCCs in chromaffin cells in a manner involving Na⁺ transport across the plasma membrane. Whether Na⁺ transport occurs via non-selective cation channels and/or through lipid nanopores remains to be determined.     

Mitochondrial Ca<Superscript>2+</Superscript> uptake pathways

Calcium (Ca²⁺) plays diverse roles in all living organisms ranging from bacteria to humans. It is a structural element for bones, an essential mediator of excitation-contraction coupling, and a universal second messenger in the regulation of ion channel, enzyme and gene expression activities. In mitochondria, Ca²⁺ is crucial for the control of energy production and cellular responses to metabolic stress. Ca²⁺ uptake by the mitochondria occurs by the uniporter mechanism. The Mitochondrial Ca2+ Uniporter (MCU) protein has recently been identified as a core component responsible for mitochondrial Ca²⁺ uptake. MCU knockout (MCU KO) studies have identified a number of important roles played by this high capacity uptake pathway. Interestingly, this work has also shown that MCU-mediated Ca²⁺ uptake is not essential for vital cell functions such as muscle contraction, energy metabolism and neurotransmission. Although mitochondrial Ca²⁺ uptake was markedly reduced, MCU KO mitochondria still contained low but detectable levels of Ca²⁺. In view of the fundamental importance of Ca²⁺ for basic cell signalling, this finding suggests the existence of other currently unrecognized pathways for Ca²⁺ entry. We review the experimental evidence for the existence of alternative Ca²⁺ influx mechanisms and propose how these mechanisms may play an integral role in mitochondrial Ca²⁺ signalling.     

Synaptically Activated Ca<Superscript>2+</Superscript> Release From Internal Stores in CNS Neurons

Synaptically activated postsynaptic [Ca²⁺]_i increases occur through three main pathways: Ca²⁺ entry through voltage-gated Ca²⁺ channels, Ca²⁺ entry through ligand-gated channels, and Ca²⁺ release from internal stores. The first two pathways have been studied intensively; release from stores has been the subject of more recent investigations.Ca²⁺ release from stores in CNS neurons primarily occurs as a result of IP₃ mobilized by activation of metabotropic glutamatergic and/or cholingergic receptors coupled to PLC. Ca²⁺ release is localized near spines in Purkinje cells and occurs as a wave in the primary apical dendrites of pyramidal cells in the hippocampus and cortex. The amplitude of the [Ca²⁺]_i increase can reach several micromolar, significantly larger than the increase due to backpropagating spikes.The large amplitude, long duration, and unique location of the [Ca²⁺]_i increases due to Ca²⁺ release from stores suggests that these increases can affect specific downstream signaling mechanisms in neurons.     

Distinctive characteristics and functions of multiple mitochondrial Ca<Superscript>2+</Superscript> influx mechanisms

Intracellular Ca²⁺ is vital for cell physiology. Disruption of Ca²⁺ homeostasis contributes to human diseases such as heart failure, neuron-degeneration, and diabetes. To ensure an effective intracellular Ca²⁺ dynamics, various Ca²⁺ transport proteins localized in different cellular regions have to work in coordination. The central role of mitochondrial Ca²⁺ transport mechanisms in responding to physiological Ca²⁺ pulses in cytosol is to take up Ca²⁺ for regulating energy production and shaping the amplitude and duration of Ca²⁺ transients in various micro-domains. Since the discovery that isolated mitochondria can take up large quantities of Ca²⁺ approximately 5 decades ago, extensive studies have been focused on the functional characterization and implication of ion channels that dictate Ca²⁺ transport across the inner mitochondrial membrane. The mitochondrial Ca²⁺ uptake sensitive to non-specific inhibitors ruthenium red and Ru360 has long been considered as the activity of mitochondrial Ca²⁺ uniporter (MCU). The general consensus is that MCU is dominantly or exclusively responsible for the mitochondrial Ca²⁺ influx. Since multiple Ca²⁺ influx mechanisms (e.g. L-, T-, and N-type Ca²⁺ channel) have their unique functions in the plasma membrane, it is plausible that mitochondrial inner membrane has more than just MCU to decode complex intracellular Ca²⁺ signaling in various cell types. During the last decade, four molecular identities related to mitochondrial Ca²⁺ influx mechanisms have been identified. These are mitochondrial ryanodine receptor, mitochondrial uncoupling proteins, LETM1 (Ca²⁺/H⁺ exchanger), and MCU and its Ca²⁺ sensing regulatory subunit MICU1. Here, we briefly review recent progress in these and other reported mitochondrial Ca²⁺ influx pathways and their differences in kinetics, Ca²⁺ dependence, and pharmacological characteristics. Their potential physiological and pathological implications are also discussed.     

CB<Subscript>1</Subscript> Receptors Mediated Inhibition of ATP-Induced [Ca<Superscript>2+</Superscript>]i Increase in Cultured Rat Spinal Dorsal Horn Neurons

Spinal cannabinoid receptor 1 (CB₁R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB₁R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB₁ receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca²⁺ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca²⁺]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100 μM ATP caused [Ca²⁺]i increase in cultured DH neurons. ATP-evoked [Ca²⁺]i increase in DH neurons was blocked by chelating extracellular Ca²⁺ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca²⁺]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca²⁺]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca²⁺ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca²⁺ response was mimicked by ACEA (CB₁R agonist), but was not influenced by AM1241 (CB₂R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca²⁺ mobilization was blocked by AM251 (CB₁ receptor antagonist), but was not influenced by AM630 (CB₂ receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB₁R rather than CB₂R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB₁R on P2X-channel-induced Ca²⁺ mobilization.     

Ca<Superscript>2+</Superscript>-Dependent Phosphorylation of RyR2 Can Uncouple Channel Gating from Direct Cytosolic Ca<Superscript>2+</Superscript> Regulation

Phosphorylation of the cardiac ryanodine receptor (RyR2) is thought to be important not only for normal cardiac excitation-contraction coupling but also in exacerbating abnormalities in Ca²⁺ homeostasis in heart failure. Linking phosphorylation to specific changes in the single-channel function of RyR2 has proved very difficult, yielding much controversy within the field. We therefore investigated the mechanistic changes that take place at the single-channel level after phosphorylating RyR2 and, in particular, the idea that PKA-dependent phosphorylation increases RyR2 sensitivity to cytosolic Ca²⁺. We show that hyperphosphorylation by exogenous PKA increases open probability (P _o) but, crucially, RyR2 becomes uncoupled from the influence of cytosolic Ca²⁺; lowering [Ca²⁺] to subactivating levels no longer closes the channels. Phosphatase (PP1) treatment reverses these gating changes, returning the channels to a Ca²⁺-sensitive mode of gating. We additionally found that cytosolic incubation with Mg²⁺/ATP in the absence of exogenously added kinase could phosphorylate RyR2 in approximately 50% of channels, thereby indicating that an endogenous kinase incorporates into the bilayer together with RyR2. Channels activated by the endogenous kinase exhibited identical changes in gating behavior to those activated by exogenous PKA, including uncoupling from the influence of cytosolic Ca²⁺. We show that the endogenous kinase is both Ca²⁺-dependent and sensitive to inhibitors of PKC. Moreover, the Ca²⁺-dependent, endogenous kinase–induced changes in RyR2 gating do not appear to be related to phosphorylation of serine-2809. Further work is required to investigate the identity and physiological role of this Ca²⁺-dependent endogenous kinase that can uncouple RyR2 gating from direct cytosolic Ca²⁺ regulation.     

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.