Store-dependent Ca2 Research Articles

АНТАГОНИСТ РЕЦЕПТОРОВ СИГМА-1, СОЕДИНЕНИЕ BD-1063, ПОДАВЛЯЕТ Са2+-ОТВЕТЫ, ВЫЗЫВАЕМЫЕ МОЛИКСАНОМ В МАКРОФАГАХ

Sigma-1 receptors are ubiquitous multifunctional ligand-operated molecular chaperones in the endoplasmic reticulum membrane with a unique history, structure, and pharmacological profile. Acting as chaperones, sigma-1 receptors modulate a wide range of cellular processes in health and disease, including Ca2+ signaling processes. The pharmacological analogue of oxidized glutathione, drug molixan®, is used as an immunomodulator and cytoprotector in the complex therapy of bacterial, viral and oncological diseases; effective in the prevention and treatment of coronavirus infection COVID-19. To elucidate the involvement of sigma-1 receptors in the effect of molixan on the intracellular Ca2+ concentration in macrophages and in the regulation of Ca2+ signaling processes in macrophages in general, the effect of the sigma-1 receptor selective antagonist, compound BD-1063, on Ca2+ responses induced by molixan in rat peritoneal macrophages was investigated. Using Fura-2AM microfluorimetry we have shown for the first time that compound BD-1063 significantly suppresses both Ca2+ mobilization from intracellular Ca2+ stores and subsequent store-dependent Ca2+ entry, induced by molixan in peritoneal macrophages. The data obtained indicate the involvement of sigma-1 receptors in the complex signaling cascade triggered by molixan and leading to intracellular Ca2+ concentration increase in macrophages. The results also suggest the involvement of sigma-1 receptors in the regulation of store-dependent Ca2+ entry in macrophages.

Tricyclic antidepressant amitriptyline attenuates Ca<sup>2+</sup> responses in rat peritoneal macrophages

Amitriptyline is a tricyclic antidepressant widely used in clinical practice for the treatment of anxiety, depression and chronic pain. These drugs have a multifaceted effect on cellular processes. One of their targets is sigma-1 receptors. Sigma-1 receptors are molecular chaperones located in endoplasmic reticulum membrane; they are characterized by a unique structure and pharmacological profile. Sigma-1 receptors regulate many cellular processes in health and disease, including Ca2+ signaling. Using Fura-2AM microfluorimetry, it was shown for the first time that sigma-1 receptor agonist, antidepressant amitriptyline, significantly suppresses both Ca2+ mobilization from intracellular Ca2+-stores and subsequent store-dependent Ca2+ entry into cells, induced by endoplasmic Ca2+-ATPase inhibitors thapsigargin and cyclopiazonic acid, as well as disulfide-containing immunomodulators glutoxim and molixan, in rat peritoneal macrophages. The results suggest the involvement of sigma-1 receptors in a complex signaling cascade induced by glutoxim or molixan, leading to an increase of intracellular Ca2+ concentration in macrophages. The results also indicate the participation of sigma-1 receptors in the regulation of store-dependent Ca2+ entry in macrophages.

Haloperidol, a sigma-1 receptor antagonist, inhibits Ca<sup>2+</sup> responses in rat peritoneal macrophages

Sigma-1 receptors are ubiquitous multifunctional ligand-operated molecular chaperones in the endoplasmic reticulum membrane and have a unique history, structure, and pharmacological profile. Sigma-1 receptors modulate a wide range of cellular processes in health and disease and Ca2+ signaling processes. Using the Ca2+ sensitive fluorescent ratiometric dye Fura-2AM, it has been shown that neuroleptic (haloperidol, an antagonist of sigma-1 receptors) significantly suppresses Ca2+ mobilization from intracellular Ca2+ stores and subsequent store-dependent Ca2+ entry, induced by thapsigargin and cyclopiazonic acid, as inhibitors of endoplasmic reticulum Ca2+-ATPases, and also by glutoxim and molixan, disulfide-containing immunomodulatory drugs, in rat peritoneal macrophages. Findings of the study revealed that sigma-1 receptors are involved in the complex signaling cascade triggered by glutoxim or molixan that leads to the increase in the intracellular Ca2+ concentration in macrophages and participate in regulation of store-dependent Ca2+ entry in macrophages.

Нейролептики подавляют Са2+-ответы, вызываемые глутоксимом и моликсаном в макрофагах

Haloperidol (butyrophenone derivative) and phenothiazine derivatives chlorpromazine and trifluoperazine belong to the typical first-generation neuroleptics and are widely used in clinical practice for treatment of schizophrenia and other mental diseases. Their multifaceted effect on cellular processes is well known. Thus, a high affinity of typical neuroleptics for sigma-1 receptors was revealed. Sigma-1 receptors are ubiquitous multifunctional ligand-operated molecular chaperones in the endoplasmic reticulum membrane with a unique history, structure, and pharmacological profile. Acting as chaperones, sigma-1 receptors modulate a wide range of cellular processes in health and disease, including Ca2+ signaling processes. To elucidate the involvement of sigma-1 receptors in Ca2+ signaling processes in macrophages, the effect of sigma-1 receptor ligands on Ca2+ responses induced by disulfide-containing immunomodulators glutoxim and molixan in rat peritoneal macrophages was investigated. Using Fura-2AM microfluorimetry we have shown that haloperidol, chlorpromazine and trifluoperazine significantly suppress both Ca2+ mobilization from intracellular Ca2+ stores and subsequent store-dependent Ca2+ entry, induced by glutoxim or molixan in peritoneal macrophages. The data obtained indicate the possible involvement of sigma-1 receptors in the complex signaling cascade triggered by glutoxim or molixan and leading to intracellular Ca2+ concentration increase in macrophages. The results also suggest the involvement of sigma-1 receptors in the regulation of store-dependent Ca2+ entry in macrophages.

Toll-like receptor 4 activation enhances Orai1-mediated calcium signal promoting cytokine production in spinal astrocytes.

Toll-like receptor 4 (TLR4) has been implicated in pathological conditions including chronic pain. Activation of astrocytic TLRs leads to the synthesis of pro-inflammatory cytokines like interleukin 6 (IL-6) and tumor necrosis factor-ɑ (TNF-α), which can cause pathological inflammation and tissue damage in the central nervous system. However, the mechanisms of TLR4-mediated cytokine releases from astrocytes are incomplete understood. Our previous study has shown that Orai1, a key component of calcium release activated calcium channels (CRACs), mediates Ca2+ entry in astrocytes. How Orai1 contributes to TLR4 signaling remains unclear. Here we show that Orai1 deficiency drastically attenuated lipopolysaccharides (LPS)-induced TNF-α and IL-6 production in astrocytes. Acute LPS treatment did not induce Ca2+ response and had no effect on thapsigargin (Ca2+-ATPase inhibitor)-induced store-dependent Ca2+ entry. Inhibition or knockdown of Orai1 showed no reduction in LPS-induced p-ERK1/2, p-c-Jun N-terminal kinase, or p-p38 MAPK activation. Interestingly, Orai1 protein level was significantly increased after LPS exposure, which was blocked by inhibition of NF-κB activity. LPS significantly increased basal Ca2+ level and SOCE after exposure to astrocytes. Moreover, elevating extracellular Ca2+ concentration increased cytosolic Ca2+ level, which was almost eliminated in Orai1 KO astrocytes. Our study reports novel findings that Orai1 acts as a Ca2+ leak channel regulating the basal Ca2+ level and enhancing cytokine production in astrocytes under the inflammatory condition. These findings highlight an important role of Orai1 in astrocytic TRL4 function and may suggest that Orai1 could be a potential therapeutic target for neuroinflammatory disorders including chronic pain.

Sigma-1 Receptor Ligands Chlorpromazine and Trifluoperazine Attenuate Ca2+ Responses in Rat Peritoneal Macrophages.

Sigma-1 receptors are ubiquitous multifunctional ligand-regulated molecular chaperones in the endoplasmic reticulum membrane with a unique history, structure, and pharmacological profile. Sigma-1 receptors bind ligands of different chemical structure and pharmacological action and modulate a wide range of cellular processes in health and disease, including Ca2+ signaling. To elucidate the involvement of sigma-1 receptors in the processes of Ca2+ signaling in macrophages we studied the effect of sigma-1 receptor ligands, phenothiazine neuroleptics chlorpromazine and trifluoperazine, on Ca2+ responses induced by inhibitors of endoplasmic Ca2+–ATPases thapsigargin and cyclopiazonic acid, as well as by disulfide-containing immunomodulators Glutoxim and Molixan in rat peritoneal macrophages. Using Fura-2AM microfluorimetry we showed for the first time that chlorpromazine and trifluoperazine inhibit both phases of Ca2+ responses induced by Glutoxim, Molixan, thapsigargin, and cyclopiazonic acid in rat peritoneal macrophages. The data obtained indicate the participation of sigma-1 receptors in a complex signaling cascade caused by Glutoxim or Molixan and leading to an increase in intracellular Ca2+ concentration in macrophages. The results also indicate the involvement of sigma-1 receptors in the regulation of store-dependent Ca2+entry in macrophages.

Injury-induced Erk1/2 signaling tissue-specifically interacts with Ca2+ activity and is necessary for regeneration of spinal cord and skeletal muscle

The transition of stem cells from quiescence to proliferation enables tissues to self-repair. The signaling mechanisms driving these stem-cell-status decisions are still unclear. Ca2+ and the extracellular signal-regulated kinase (Erk1/2) are two signaling pathways that have the potential to coordinate multiple signals to promote a specific cellular response. They both play important roles during nervous system development but their roles during spinal cord and muscle regeneration are not fully deciphered. Here we show in Xenopus laevis larvae that both Ca2+ and Erk1/2 signaling pathways are activated after tail amputation. In response to injury, we find that Erk1/2 signaling is activated in neural and muscle stem cells and is necessary for spinal cord and skeletal muscle regeneration. Finally, we show in vivo that Erk1/2 activity is necessary for an injury-induced increase in intracellular store-dependent Ca2+ dynamics in skeletal muscle-associated tissues but that in spinal cord, injury increases Ca2+ influx-dependent Ca2+ activity independent of Erk1/2 signaling. This study suggests that precise temporal and tissue-specific activation of Ca2+ and Erk1/2 pathways is essential for regulating spinal cord and muscle regeneration.

Manipulations of Glutathione Metabolism Modulate IP3-Mediated Store-Operated Ca2+ Entry on Astroglioma Cell Line.

The regulation of the redox status involves the activation of intracellular pathways as Nrf2 which provides hormetic adaptations against oxidative stress in response to environmental stimuli. In the brain, Nrf2 activation upregulates the formation of glutathione (GSH) which is the primary antioxidant system mainly produced by astrocytes. Astrocytes have also been shown to be themselves the target of oxidative stress. However, how changes in the redox status itself could impact the intracellular Ca2+ homeostasis in astrocytes is not known, although this could be of great help to understand the neuronal damage caused by oxidative stress. Indeed, intracellular Ca2+ changes in astrocytes are crucial for their regulatory actions on neuronal networks. We have manipulated GSH concentration in astroglioma cells with selective inhibitors and activators of the enzymes involved in the GSH cycle and analyzed how this could modify Ca2+ homeostasis. IP3-mediated store-operated calcium entry (SOCE), obtained after store depletion elicited by Gq-linked purinergic P2Y receptors activation, are either sensitized or desensitized, following GSH depletion or increase, respectively. The desensitization may involve decreased expression of the proteins STIM2, Orai1, and Orai3 which support SOCE mechanism. The sensitization process revealed by exposing cells to oxidative stress likely involves the increase in the activity of Calcium Release-Activated Channels (CRAC) and/or in their membrane expression. In addition, we observe that GSH depletion drastically impacts P2Y receptor-mediated changes in membrane currents, as evidenced by large increases in Ca2+-dependent K+ currents. We conclude that changes in the redox status of astrocytes could dramatically modify Ca2+ responses to Gq-linked GPCR activation in both directions, by impacting store-dependent Ca2+-channels, and thus modify cellular excitability under purinergic stimulation.

Differential activation of Ca2+ influx channels modulate stem cell potency, their proliferation/viability and tissue regeneration

Stem cells have indefinite self-renewable capability; however, factors that modulate their pluripotency/function are not fully identified. Here we show that store-dependent Ca2+ entry is essential for modulating the function of bone marrow-derived mesenchymal stem cells (MSCs). Increasing external Ca2+ modulated cell cycle progression that was critical for MSCs survival. Additionally, Ca2+ was critical for stem proliferation, its differentiation, and maintaining stem cell potential. Ca2+ channel characterization, including gene silencing, showed two distinct Ca2+ entry channels (through Orai1/TRPC1 or via Orai3) that differentially regulate the proliferation and viability of MSCs. Importantly, NFκB translocation, but not JNK/ERK into the nucleus, was observed upon store depletion, which was blocked by the addition of Ca2+ channel inhibitors. Radiation lead to a decrease in saliva secretion, decrease in acinar cell number, and enlarged ducts were observed, which were restored by the transplantation of stem cells that were propagated in higher Ca2+. Finally radiation showed a decrese in TRPC1 expression along with a decrese in AQP5, which was again restored upon MSC tranplantation. Together these results suggest that Ca2+ entry is essential for stem cell function that could be critical for regenerative medicine.

Store-Independent Calcium Entry and Related Signaling Pathways in Breast Cancer.

Known as a key effector in breast cancer (BC) progression, calcium (Ca2+) is tightly regulated to maintain the desired concentration to fine-tune cell functions. Ca2+ channels are the main actors among Ca2+ transporters that control the intracellular Ca2+ concentration in cells. It is well known that the basal Ca2+ concentration is regulated by both store-dependent and independent Ca2+ channels in BC development and progression. However, most of the literature has reported the role of store-dependent Ca2+ entry, and only a few studies are focusing on store-independent Ca2+ entry (SICE). In this review, we aim to summarize all findings on SICE in the BC progression field.

Sigma1 Receptor Inhibits TRPC1-Mediated Ca2+ Entry That Promotes Dopaminergic Cell Death.

Regulation of Ca2+ homeostasis is essential for neuronal function and its survival. Recent data suggest that TRPC1 function as the endogenous store-mediated Ca2+ entry channel in dopaminergic cells, and loss of TRPC1 function leads to neurodegeneration; however, its regulation is not fully identified. Here we provide evidence that the sigma 1 receptor contributes to the loss of dopaminergic cells by blocking TRPC1-mediated Ca2+ entry. Importantly, downregulation of sigma 1 receptor expression significantly decreased neurotoxin-induced loss of dopaminergic cells as measured by MTT assays and caspase activity was also inhibited. Importantly, sigma 1 receptor inhibited TRPC1-mediated Ca2+ entry and silencing of sigma 1 receptor significantly restored store-dependent Ca2+ influx. Although co-immunoprecipitation failed to show an interaction between the TRPC1 and sigma 1 receptor, store depletion promoted a decrease in the sigma 1 receptor-STIM1 association. Neurotoxin-induced loss of Ca2+ entry was significantly restored in cells that had decreased sigma 1 receptor expression. Furthermore, TRPC1 or STIM1 silencing inhibited store-mediated Ca2+ entry, which was further increased upon the downregulation of the sigma 1 receptor expression. TRPC1 silencing prevented the increased neuroprotection and caspase activity observed upon the downregulation of sigma 1 receptor. Finally, sigma 1 receptor activation also significantly decreased TRPC1-mediated Ca2+ entry and lead to an increase in neurodegeneration. In contrast, addition of sigma 1 receptor antagonist prevented neurotoxin-induced neurodegeneration and facilitated TRPC1-mediated Ca2+ influx. Together these results suggest that the sigma 1 receptor is involved in the inhibition of TRPC1- mediated Ca2+ entry, which leads to the degeneration in the dopaminergic cells, and prevention of sigma 1 receptor function could protect neuronal cell death as observed in Parkinson's disease.

Neuroleptic Chlorpromazine Modulates Ca2+ Responses in Macrophages.

Using Fura-2AM microfluorimetry, we have shown for the first time that sigma-1 receptor antagonist neuroleptic chlorpromazine significantly inhibits glutoxim- and molixan-induced Ca2+ responses and Ca2+ responses induced by endoplasmic reticulum Са2+-ATPase inhibitors thapsigargin and cyclopiazonic acid in rat peritoneal macrophages. The results suggest the involvement of sigma-1 receptors in the signaling cascade induced by glutoxim or molixan and leading to intracellular Ca2+ concentration increase and in the regulation of store-dependent Ca2+ entry in macrophages.

Loss of Ca2+ entry via Orai-TRPC1 induces ER stress, initiating immune activation in macrophages.

Activation of cellular stresses is associated with inflammation; however, the mechanisms are not well identified. Here, we provide evidence that loss of Ca2+ influx induces endoplasmic reticulum (ER) stress in primary macrophages and in murine macrophage cell line Raw 264.7, in which the unfolded protein response is initiated to modulate cytokine production, thereby activating the immune response. Stressors that initiate the ER stress response block store-dependent Ca2+ entry in macrophages prior to the activation of the unfolded protein response. The endogenous Ca2+ entry channel is dependent on the Orai1-TRPC1-STIM1 complex, and the presence of ER stressors decreased expression of TRPC1, Orai1 and STIM1. Additionally, blocking Ca2+ entry with SKF96365 also induced ER stress, promoted cytokine production, activation of autophagy, increased caspase activation and induced apoptosis. Furthermore, ER stress inducers inhibited cell cycle progression, promoted the inflammatory M1 phenotype, and increased phagocytosis. Mechanistically, restoration of Orai1-STIM1 expression inhibited the ER stress-mediated loss of Ca2+ entry that prevents ER stress and inhibits cytokine production, and thus induced cell survival. These results suggest an unequivocal role of Ca2+ entry in modulating ER stress and in the induction of inflammation.

Sigma-1 receptor agonist amitriptyline inhibits store-dependent Ca2+ entry in macrophages

Using Fura-2AM microfluorimetry we have shown for the first time that sigma-1 receptor agonist - tricyclic antidepressant amitriptyline - significantly inhibits store-dependent Ca2+ entry, induced by endoplasmic Ca2+-ATPase inhibitors thapsigargin and cyclopiazonic acid, in rat peritoneal macrophages. The results suggest possible involvement of sigma-1 receptors in the regulation of store-dependent Ca2+ entry in macrophages.

Sigma-1 Receptor Agonist Amitriptyline Inhibits Store-Dependent Ca2+ Entry in Macrophages.

Using Fura-2AM microfluorimetry, we have shown for the first time that sigma-1 receptor agonist-tricyclic antidepressant amitriptyline-significantly inhibits store-dependent Ca2+ entry, induced by endoplasmic Ca2+-ATPase inhibitors thapsigargin and cyclopiazonic acid, in rat peritoneal macrophages. The results suggest a possible involvement of sigma-1 receptors in the regulation of store-dependent Ca2+ entry in macrophages.

Septins organize endoplasmic reticulum-plasma membrane junctions for STIM1-ORAI1 calcium signalling

ORAI1 Ca2+ channels in the plasma membrane (PM) are gated by STIM1 at endoplasmic reticulum (ER)-PM junctions to effect store-dependent Ca2+ entry into cells, but little is known about how local STIM-ORAI signalling at junctions is coordinated with overall cellular architecture. Filamentous septins can specify cytoskeletal rearrangements and have been found recently to modulate STIM-ORAI signalling. Here we show by super-resolution imaging of ORAI1, STIM1, and septin 4 in living cells that septins facilitate Ca2+ signalling indirectly. Septin 4 does not colocalize preferentially with ORAI1 in resting or stimulated cells, assemble stably at ER-PM junctions, or specify a boundary that directs or confines ORAI1 to junctions. Rather, ORAI1 is recruited to junctions solely through interaction with STIM proteins, while septins regulate the number of ER-PM junctions and enhance STIM1-ORAI1 interactions within junctions. Thus septins communicate with STIM1 and ORAI1 through protein or lipid intermediaries, and are favorably positioned to coordinate Ca2+ signalling with rearrangements in cellular architecture.

Sigma-1 Receptor Antagonist Haloperidol Attenuates Store-Dependent Ca2+ 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.

The role of proteins Stim and Orai as molecular components of the store-dependent current Ca2+ in lymphocytes

In the process of evolution of eukaryotes has formatted a highly organized mechanism for maintaining and regulating intracellular calcium homeostasis, which is one of the most important components of cell signaling in all branches of the phylogenetic tree. Intracellular calcium controls numerous physiological processes in the cell. Ca2+ forms signals as their spatial-temporal distribution. The frequency and amplitude of calcium oscillations depends on the signal strength. Calcium signals causing long-term or short-term responses of cells. Mainly, calcium signals in lymphocytes mediate gene expression program initiation that leads to proliferation, differentiation and production of proinflammatory cytokines also activate formation of inflammasome. Therefore, calcium signals mediate immune, and inflammatory response, autoimmune reaction of lymphocytes. The main mechanism of calcium signaling in lymphocytes is store-dependent Ca2+ current. Mobilization of cellular Ca2+ in response to receptor stimulation commonly occurs through release of Ca2+ ions from intracellular Ca2+ stores or influx across the plasma membrane through calcium - selective channels. Calciumselective channels are assembled from two protein families: the Orai proteins which form the ion channel pore, and the stromal interaction molecule (STIM) proteins which function as endoplasmic reticulum calcium sensors and activators of the channel. Stim protein is a transmembrane monomer which is localized at the membrane of the endoplasmic reticulum. This molecule is a sensor Ca2+ in response to emptying store activates calciumselective channels the plasma membrane. These channels express proteins Orai which are tetramers forming inside the channel pore and act as a site Ca2+. Orai binds to Stim. Orai proteins are activated after receiving information from Stim about Store depletion. Thus, the relationship and coordination of Stim and Orai proteins provides store - dependent Ca2+ current and causes cellular functional responses. Increased Ca2+ levels induce the activation of transcription factors such as NFAT, JNK1, MEF2, CREB, and, in most cases, is a crucial factor in the all differentiation or death. In this review, the mechanism of the store-dependent Ca2+ current in lymphocytes is presented.

Arachidonic acid-induced Ca2+ entry and migration in a neuroendocrine cancer cell line

BackgroundStore-operated Ca2+ entry (SOCE) has been implicated in the migration of some cancer cell lines. The canonical SOCE is defined as the Ca2+ entry that occurs in response to near-maximal depletion of Ca2+ within the endoplasmic reticulum. Alternatively, arachidonic acid (AA) has been shown to induce Ca2+ entry in a store-independent manner through Orai1/Orai3 hetero-multimeric channels. However, the role of this AA-induced Ca2+ entry pathway in cancer cell migration has not been adequately assessed.MethodsThe present study investigated the involvement of AA-induced Ca2+ entry in migration in BON cells, a model gastro-enteropancreatic neuroendocrine tumor (GEPNET) cell line using pharmacological and gene knockdown methods in combination with live cell fluorescence imaging and standard migration assays.ResultsWe showed that both the store-dependent and AA-induced Ca2+ entry modes could be selectively activated and that exogenous administration of AA resulted in Ca2+ entry that was pharmacologically distinct from SOCE. Also, whereas homomeric Orai1-containing channels appeared to largely underlie SOCE, the AA-induced Ca2+ entry channel required the expression of Orai3 as well as Orai1. Moreover, we showed that AA treatment enhanced the migration of BON cells and that this migration could be abrogated by selective inhibition of the AA-induced Ca2+ entry.ConclusionsTaken together, these data revealed that an alternative Orai3-dependent Ca2+ entry pathway is an important signal for GEPNET cell migration.

Trifluoperazine Attenuates Store-Dependent Ca2+ 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.