Presence Of Extracellular Ca2 Research Articles

Insights into the mediation of Ca2+ signaling in the promoting effects of LETX-VI on the synthesis and release of dopamine.

Latroeggtoxin-VI (LETX-VI) is an active protein and was previously demonstrated to have effects on the synthesis and release of dopamine. Hererin, the involvement of Ca2+ signaling in the effects of LETX-VI on dopamine was systematically investigated, using PC12 cells as a neuron model. LETX-VI was shown to promote dopamine release from PC12 cells both in the presence and absence of extracellular Ca2+; however the presence of extracellular Ca2+ was favorable for enhancing the promoting effects of LETX-VI on dopamine, because LETX-VI facilitated the influx of extracellular Ca2+ through the L-type calcium channels in plasma membrane (PM) to increase cytosolic Ca2+ concentration. LETX-VI was able to penetrate the PM of PC12 cells to act on the Ca2+ channel proteins IP3Rs and RyRs in the endoplasm reticulum (ER) membrane, opening the Ca2+ channels and promoting the release of ER Ca2+ to elevate cytosolic Ca2+ level. With the help of intracellular Ca2+ chelator BAPTA, the elevated cytosolic Ca2+ level was proven to play crucial role for the enhanced promoting effects of LETX-VI on dopamine. Taken together, LETX-VI is able to open the Ca2+ channels in both PM and ER membrane simultaneously to facilitate extracellular Ca2+ influx and ER Ca2+ release, and thus increases the cytosolic Ca2+ concentration to enhance the promoting effects on the synthesis and release of dopamine.

Functional Expression of IP, 5-HT4, D1, A2A, and VIP Receptors in Human Odontoblast Cell Line.

Odontoblasts are involved in sensory generation as sensory receptor cells and in dentin formation. We previously reported that an increase in intracellular cAMP levels by cannabinoid 1 receptor activation induces Ca2+ influx via transient receptor potential vanilloid subfamily member 1 channels in odontoblasts, indicating that intracellular cAMP/Ca2+ signal coupling is involved in dentinal pain generation and reactionary dentin formation. Here, intracellular cAMP dynamics in cultured human odontoblasts were investigated to understand the detailed expression patterns of the intracellular cAMP signaling pathway activated by the Gs protein-coupled receptor and to clarify its role in cellular functions. The presence of plasma membrane Gαs as well as prostaglandin I2 (IP), 5-hydroxytryptamine 5-HT4 (5-HT4), dopamine D1 (D1), adenosine A2A (A2A), and vasoactive intestinal polypeptide (VIP) receptor immunoreactivity was observed in human odontoblasts. In the presence of extracellular Ca2+, the application of agonists for the IP (beraprost), 5-HT4 (BIMU8), D1 (SKF83959), A2A (PSB0777), and VIP (VIP) receptors increased intracellular cAMP levels. This increase in cAMP levels was inhibited by the application of the adenylyl cyclase (AC) inhibitor SQ22536 and each receptor antagonist, dose-dependently. These results suggested that odontoblasts express Gs protein-coupled IP, 5-HT4, D1, A2A, and VIP receptors. In addition, activation of these receptors increased intracellular cAMP levels by activating AC in odontoblasts.

Searching for Mechanisms Underlying the Assembly of Calcium Entry Units: The Role of Temperature and pH

Store-operated Ca2+ entry (SOCE) is a mechanism that allows muscle fibers to recover external Ca2+, which first enters the cytoplasm and then, via SERCA pump, also refills the depleted intracellular stores (i.e., the sarcoplasmic reticulum, SR). We recently discovered that SOCE is mediated by Calcium Entry Units (CEUs), intracellular junctions formed by: (i) SR stacks containing STIM1; and (ii) I-band extensions of the transverse tubule (TT) containing Orai1. The number and size of CEUs increase during prolonged muscle activity, though the mechanisms underlying exercise-dependent formation of new CEUs remain to be elucidated. Here, we first subjected isolated extensor digitorum longus (EDL) muscles from wild type mice to an ex vivo exercise protocol and verified that functional CEUs can assemble also in the absence of blood supply and innervation. Then, we evaluated whether parameters that are influenced by exercise, such as temperature and pH, may influence the assembly of CEUs. Results collected indicate that higher temperature (36 °C vs. 25 °C) and lower pH (7.2 vs. 7.4) increase the percentage of fibers containing SR stacks, the n. of SR stacks/area, and the elongation of TTs at the I band. Functionally, assembly of CEUs at higher temperature (36 °C) or at lower pH (7.2) correlates with increased fatigue resistance of EDL muscles in the presence of extracellular Ca2+. Taken together, these results indicate that CEUs can assemble in isolated EDL muscles and that temperature and pH are two of the possible regulators of CEU formation.

Calcium signaling of primary chondrocytes and ATDC5 chondrogenic cells under osmotic stress and mechanical stimulation

Calcium signaling plays an essential role in chondrocyte mechanotransduction. Guilak and colleagues have revealed the roles of TRPV4 and Piezo channels in chondrocyte calcium signaling and metabolism. This study compared the calcium responses of primary chondrocytes and ATDC5 cells induced by two different stimuli: osmotic stress and intense mechanical stimulus. Roles of three essential calcium signaling pathways, including extracellular calcium source, intracellular ER calcium store and mechanical-sensitive ion channels, were also investigated and compared between cells. Primary chondrocytes showed more vigorous calcium peaks under osmotic stress than under mechanical stimuli, while an opposite trend was found for ATDC5 cells. Extracellular calcium source, intracellular ER store, and PLC/IP3 pathway each played significant roles in the calcium responses of ATDC5 cells under both osmotic and mechanical stimuli. However, high level shear stress can directly cause ER release in primary cells without the presence of extracellular Ca2+ or involvement of PLC-IP3 pathway. TRPV4 channel is essential for the responses of ATDC5 cells, but not for primary chondrocytes. In contrast, inhibition of mechano-sensitive channels had no significant effects on the ATDC5 cells. Therefore, primary chondrocytes and ATDC5 cells rely on distinct calcium sources and ion channels to initiate intracellular calcium signaling. Together, these results contribute to our understanding of stimulation-induced calcium signaling in primary chondrocytes and ATDC5 cells, and the different roles of three essential pathways between the two cell types.

Periventricular leukomalaci (PVL) is critically dependent upon the presence of extracellular ions (Ca2+ and Na+)

This work is clarifying the role of extracellular ions in PVL in neonates. Ischaemic injury of adult brainwas critically dependent upon the presence of extracellular Ca²+ as revealed by a range of studies (Styset al., 1992). Along with they found that two extracellular ions were essential for the development ofpermanent loss of function during ischaemia in adult brain: Ca²⁺ and Na⁺. The dependence of ischaemicinjury in adult upon both Ca²⁺ and Na⁺ indicates that these ions are potentially toxic to cell viability bycausing permanent loss to axonal function. Therefore, the effects of these two ions during 90 minoxygen-glucose deprivation (OGD), (the practical model of ischaemia) in neonatal brain wereinvestigated using electrophysiology techniques for compound action potential (CAP) recording. Thisproduced the interesting finding that removal of either Ca²⁺ or Na⁺ from the extracellular spaceprotected the neonatal brain from ischaemic injury during OGD. When Ca²⁺ or Na⁺ were removed fromthe extracellular space during 90 min OGD, the CAP declined at the end of OGD. Afterward, the CAPrecovered following restoration of normal conditions. The results of this study indicated that,extracellular Ca²⁺ and Na⁺ are essential for the development of permanent loss of function during OGDin neonatal brain.

Possible mechanisms underlying the dynamic assembly of calcium entry units: The role of temperature and pH

Skeletal muscle function is regulated by intracellular Ca2+ levels. Two main mechanisms control movements of Ca2+ ions from intracellular stores (i.e., the sarcoplasmic reticulum; SR) and from extracellular space: (1) excitation-contraction (EC) coupling and (2) store-operated Ca2+ entry (SOCE). SOCE allows recovery of extracellular Ca2+ during prolonged muscle activity, when the SR undergoes depletion. We recently discovered that prolonged exercise leads to formation of calcium entry units (CEUs), intracellular junctions located at the I band that are formed by two distinct elements: SR stacks and transverse tubules (TTs). Assembly of CEUs during exercise promotes the interaction between STIM1 and Orai1, the two main proteins that mediate SOCE, and increases muscle resistance to fatigue in the presence of extracellular Ca2+. The molecular mechanisms underlying the exercise-dependent remodeling of SR and TT leading to CEU assembly remain to be fully elucidated. Here, we first verified whether CEUs can assemble ex vivo (in the absence of blood supply and innervation), subjecting excised EDL muscles from mice to an ex vivo incremental fatigue protocol (80 Hz tetanus stimulation lasting 45 min): the data collected demonstrate that CEUs can assemble ex vivo in isolated EDL muscles. We then evaluated if intracellular parameters that are affected by exercise, such as temperature and pH, may influence the assembly of CEUs. We found that higher temperature (36°C versus 25°C) and lower pH (7.2 versus 7.4) promotes formation of CEUs increasing the percentage of fibers containing SR stacks, the number of SR stacks/area, and the elongation of TTs at the I band. Importantly, increased assembly of CEUs at higher temperature (36°C) or at lower pH (7.2) correlated with increased fatigue resistance of EDL muscles in the presence of extracellular Ca2+, suggesting that CEUs assembled ex vivo are functional.

Bi-component HlgC/HlgB and HlgA/HlgB γ-hemolysins from S. aureus: Modulation of Ca2+ channels activity through a differential mechanism

Staphylococcal bi-component leukotoxins known as *pore-forming toxins* induce upon a specific binding to membrane receptors, two independent cellular events in human neutrophils. First, they provoke the opening of pre-existing specific ionic channels including Ca2+ channels. Then, they form membrane pores specific to monovalent cations leading to immune cells death. Among these leukotoxins, HlgC/HlgB and HlgA/HlgB γ-hemolysins do act in synergy to induce the opening of different types of Ca2+ channels in the absence as in the presence of extracellular Ca2+. Here, we investigate the mechanism underlying the modulation of Ca2+-independent Ca2+ channels in response to both active leukotoxins in human neutrophils. In the absence of extracellular Ca2+, the Mn2+ has been used as a Ca2+ surrogate to determine the activity of Ca2+-independent Ca2+ channels. Our findings provide new insights about different mechanisms involved in the staphylococcal γ-hemolysins activity to regulate three different types of Ca2+-independent Ca2+ channels. We conclude that (i) HlgC/HlgB stimulates the opening of La3+-sensitive Ca2+ channels, through a cholera toxin-sensitive G protein, (ii) HlgA/HlgB stimulates the opening of Ca2+ channels not sensitive to La3+, through a G protein-independent process, and (iii) unlike HlgA/HlgB, HlgC/HlgB toxins prevent the opening of a new type of Ca2+ channels by phosphorylation/de-phosphorylation-dependent mechanisms.

Effects of high-frequency nanosecond pulses on prostate cancer cells

Electroporation with pulsed electric fields show a potential to be applied as an experimental focal therapy of tumors. Sub-microsecond regime of electric pulses displays unique electrophysical features operative in cells and membranes. Recently, MHz compression of nanosecond pulses electric fields (nsPEFs) bursts proved to enhance the effectiveness of the therapy. High morbidity of prostate cancer (PCa) and risk of overtreatment associated with this malignancy call for new minimal-invasive treatment alternative. Herein we present the in vitro study for developing applications based on this new technology. In this study, we used flow cytometric analysis, cell viability assay, caspase activity analysis, wound healing assay, confocal microscopy study, and immunofluorescence to investigate the biological effect of high-frequency nsPEFs on PCa cells. Our results show that high-frequency nsPEFs induces the permeabilization and cell death of PCa cells. The cytotoxicity is significantly enhanced in MHz compression of pulses and with the presence of extracellular Ca2+. High-frequency nsPEFs trigger changes in PCa cells’ cytoskeleton and their mobility. The presented data show a therapeutic potential of high-frequency nsPEFs in a PCa setting. The sub-microsecond regime of pulses can potentially be applied in nanosecond electroporation protocols for PCa treatment.

Ascorbate-induced oxidative stress mediates TRP channel activation and cytotoxicity in human etoposide-sensitive and -resistant retinoblastoma cells

AbstractThere are indications that pharmacological doses of ascorbate (Asc) used as an adjuvant improve the chemotherapeutic management of cancer. This favorable outcome stems from its cytotoxic effects due to prooxidative mechanisms. Since regulation of intracellular Ca2+ levels contributes to the maintenance of cell viability, we hypothesized that one of the effects of Asc includes disrupting regulation of intracellular Ca2+ homeostasis. Accordingly, we determined if Asc induced intracellular Ca2+ influx through activation of pertussis sensitive Gi/o-coupled GPCR which in turn activated transient receptor potential (TRP) channels in both etoposide-resistant and -sensitive retinoblastoma (WERI-Rb1) tumor cells. Ca2+ imaging, whole-cell patch-clamping, and quantitative real-time PCR (qRT-PCR) were performed in parallel with measurements of RB cell survival using Trypan Blue cell dye exclusion. TRPM7 gene expression levels were similar in both cell lines whereas TRPV1, TRPM2, TRPA1, TRPC5, TRPV4, and TRPM8 gene expression levels were downregulated in the etoposide-resistant WERI-Rb1 cells. In the presence of extracellular Ca2+, 1 mM Asc induced larger intracellular Ca2+ transients in the etoposide-resistant WERI-Rb1 than in their etoposide-sensitive counterpart. With either 100 µM CPZ, 500 µM La3+, 10 mM NAC, or 100 µM 2-APB, these Ca2+ transients were markedly diminished. These inhibitors also had corresponding inhibitory effects on Asc-induced rises in whole-cell currents. Pertussis toxin (PTX) preincubation blocked rises in Ca2+ influx. Microscopic analyses showed that after 4 days of exposure to 1 mM Asc cell viability fell by nearly 100% in both RB cell lines. Taken together, one of the effects underlying oxidative mediated Asc-induced WERI-Rb1 cytotoxicity stems from its promotion of Gi/o coupled GPCR mediated increases in intracellular Ca2+ influx through TRP channels. Therefore, designing drugs targeting TRP channel modulation may be a viable approach to increase the efficacy of chemotherapeutic treatment of RB. Furthermore, Asc may be indicated as a possible supportive agent in anti-cancer therapies.Ascorbate can act as an oxidant to induce tumor cell death at a pharmacological dose. Here the authors show that this response is associated with increases in GPCR Gi/o activity. This effect promotes rises in intracellular Ca2+ influx through transient receptor potential channel activity in retinoblastoma cells.

Small intestinal glucose and sodium absorption through calcium‐induced calcium release and store‐operated Ca2+ entry mechanisms

Background and PurposeLuminal glucose enhances intestinal Ca2+ absorption through apical Cav1.3 channels necessary for GLUT2‐mediated glucose absorption. As these reciprocal mechanisms are not well understood, we investigated the regulatory mechanisms of intestinal [Ca2+]cyt and SGLT1‐mediated Na+‐glucose co‐transports.Experimental ApproachGlucose absorption and channel expression were examined in mouse upper jejunal epithelium using an Ussing chamber, immunocytochemistry and Ca2+ and Na+ imaging in single intestinal epithelial cells.Key ResultsGlucose induced jejunal Isc via Na+‐glucose cotransporter 1 (SGLT1) operated more efficiently in the presence of extracellular Ca2+. A crosstalk between luminal Ca2+ entry via plasma Cav1.3 channels and the ER Ca2+ release through ryanodine receptor (RYR) activation in small intestinal epithelial cell (IEC) or Ca2+‐induced Ca2+ release (CICR) mechanism was involve in Ca2+‐mediated jejunal glucose absorption. The ER Ca2+ release through RyR triggered basolateral Ca2+ entry or store‐operated Ca2+ entry (SOCE) mechanism and the subsequent Ca2+ entry via Na+/Ca2+ exchanger 1 (NCX1) were found to be critical in Na+‐glucose cotransporter‐mediated glucose absorption. Blocking RyR, SOCE and NCX1 inhibited glucose induced [Na+]cyt and [Ca2+]cyt in single IEC and protein expression and co‐localization of STIM1/Orai1, RyR1 and NCX1 were detected in IEC and jejunal mucosa.Conclusion and ImplicationsLuminal Ca2+ influx through Cav1.3 triggers the CICR through RyR1 to deplete the ER Ca2+, which induces the basolateral STIM1/Orai1‐mediated SOCE mechanism and the subsequent Ca2+ entry via NCX1 to regulate intestinal glucose uptake via Ca2+ signalling. Targeting these mechanisms in IEC may help to modulate blood glucose and sodium in the metabolic disease.

P2Y2 and P2X4 Receptors Mediate Ca2+ Mobilization in DH82 Canine Macrophage Cells.

Purinergic receptors of the P2 subclass are commonly found in human and rodent macrophages where they can be activated by adenosine 5′-triphosphate (ATP) or uridine 5′-triphosphate (UTP) to mediate Ca2+ mobilization, resulting in downstream signalling to promote inflammation and pain. However, little is understood regarding these receptors in canine macrophages. To establish a macrophage model of canine P2 receptor signalling, the expression of these receptors in the DH82 canine macrophage cell line was determined by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. P2 receptor function in DH82 cells was pharmacologically characterised using nucleotide-induced measurements of Fura-2 AM-bound intracellular Ca2+. RT-PCR revealed predominant expression of P2X4 receptors, while immunocytochemistry confirmed predominant expression of P2Y2 receptors, with low levels of P2X4 receptor expression. ATP and UTP induced robust Ca2+ responses in the absence or presence of extracellular Ca2+. ATP-induced responses were only partially inhibited by the P2X4 receptor antagonists, 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP), paroxetine and 5-BDBD, but were strongly potentiated by ivermectin. UTP-induced responses were near completely inhibited by the P2Y2 receptor antagonists, suramin and AR-C118925. P2Y2 receptor-mediated Ca2+ mobilization was inhibited by U-73122 and 2-aminoethoxydiphenyl borate (2-APB), indicating P2Y2 receptor coupling to the phospholipase C and inositol triphosphate signal transduction pathway. Together this data demonstrates, for the first time, the expression of functional P2 receptors in DH82 canine macrophage cells and identifies a potential cell model for studying macrophage-mediated purinergic signalling in inflammation and pain in dogs.

Continuous Assembly of β-Roll Structures Is Implicated in the Type I-Dependent Secretion of Large Repeat-in-Toxins (RTX) Proteins

Repeats-in-Toxin (RTX) proteins of Gram-negative bacteria are excreted through the type I secretion system (T1SS) that recognizes non-cleavable C-terminal secretion signals. These are preceded by arrays of glycine and aspartate-rich nonapeptide repeats grouped by four to eight β strands into blocks that fold into calcium-binding parallel β-roll structures. The β-rolls are interspersed by linkers of variable length and sequence and the organization of multiple RTX repeat blocks within large RTX domains remains unknown. Here we examined the structure and function of the RTX domain of Bordetella pertussis adenylate cyclase toxin (CyaA) that is composed of five β-roll RTX blocks. We show that the non-folded RTX repeats maintain the stability of the CyaA polypeptide in the Ca2+-depleted bacterial cytosol and thereby enable its efficient translocation through the T1SS apparatus. The efficacy of secretion of truncated CyaA constructs was dictated by the number of retained RTX repeat blocks and depended on the presence of extracellular Ca2+ ions. We further describe the crystal structure of the RTX blocks IV–V of CyaA (CyaA1372–1681) that consists of a contiguous assembly of two β-rolls that differs substantially from the arrangement of the RTX blocks observed in RTX lipases or other RTX proteins. These results provide a novel structural insight into the architecture of the RTX domains of large RTX proteins and support the “push-ratchet” mechanism of the T1SS-mediated secretion of very large RTX proteins.

Bi-component Staphylococcal Leukotoxins Induce Chloride Ions Fluxes in Human Neutrophils: Opening of Ca<sup>2+</sup>-activated Cl<sup>-</sup> Channels

The bi-component leukotoxins; γ-hemolysin and Panton and Valentin Leukocidin (PVL) from Staphylococcus aureus induce two independent cellular events 1) the formation of trans-membrane pores not permeable to chloride (Cl-) ions and 2) the activation of at least, two modes of chloride fluxes (efflux/influx), including pre-existing Ca2+-activated Cl- channels (CaCC) in human polymorphonuclear neutrophils (PMNs). This was investigated by using spectrofluorometry techniques and the chloride-sensitive quencher fluorescent indicator, MQAE (N-(6-methoxyquinolyl) acetoacethyl ester). The ethidium bromide was used as an indicator for the trans-membrane pores formation by staphylococcal leukotoxins. In the absence of extracellular Ca2+, HlgA/HlgB, HlgC/HlgB and LukS-PV/LukF-PV leukotoxins from S. aureus induced a massive efflux of chloride (Cl-) ions. Interestingly, in the presence of extracellular Ca2+, the HlgA/HlgB γ-hemolysin provoked a biphasic response of Cl- movements (efflux/influx). Conversely to HlgA/HlgB and LukS-PV/LukF-PV, HlgC/HlgB leukotoxins did not induce any Cl- movement under this condition (e.g. in the presence of extracellular Ca2+). The potent Cl- channel inhibitor, DIDS, did inhibit significantly the Cl- fluxes caused by all pairs of staphylococcal leukotoxins tested in both conditions. In the present study, we found that the inhibitory effect of flufenamic acid, known as a Cl- channel inhibitor, was restricted only to the Ca2+-dependent Cl- influx triggered only by HlgA/HlgB and LukS-PV/LukF-PV leukotoxins. These findings might suggest that, Cl- fluxes in human neutrophils did involve at least, two different types of Cl- pathways, depending on the absence or presence of extracellular Ca2+. Both Cl- channels blockers, DIDS and flufenamic acid did not alter the pores formation by staphylococcal leukotoxins. Furthermore, under conditions when the membrane pores formation was blocked by divalent ions (Ca2+ and/or Zn2+), Cl- ions movements were still observed. Taken together, our results strongly provide an evidence that: i) trans-membrane pores formed by staphylococcal leukotoxins: HlgA/HlgB, HlgC/HlgB (γ-hemolysin) and LukS-PV/LukF-PV (PVL) do not drive Cl- ions fluxes ii) at least, two different types of Cl- ions pathways are activated, depending on the absence or presence of extracellular Ca2+, including Ca2+-activated Cl- channels (CaCC) and, iii) Ca2+-activated Cl- channels are mediated only by HlgA/HlgB and LukS-PV/LukF-PV leukotoxins.

Staphylococcal Pore-forming Leukotoxins: Opening of Ca<sup>2+</sup>-activated K<sup>+</sup> Channels and Specificity of Membrane Pores in Human Neutrophils

Pore-forming toxins are key virulence determinants produced by human bacterial pathogens Staphylococcus aureus, inducing two independent cellular events in neutrophils. Upon a specific binding to membrane receptors, both Panton and Valentin Leukocidin and γ-hemolysin induced an increase of Na⁺ and K⁺ fluxes, likely associated to the activation of preexisting ionic channels or to the membrane pores formation. This was investigated by using, spectrofluorometry techniques and, specific molecular probes in human neutrophils. Interestingly, we found that, in the absence of extracellular Ca²⁺, leukotoxins did form membrane pores, which were large enough to allow a massive entry of ethidium into neutrophils. Simultaneously, sustained Na⁺ influx and K⁺ efflux were observed. Another set of experiments carried out in the presence of extracellular Ca²⁺ did show that, the percentage of pores formed by leukotoxins was significantly, reduced due to the Ca²⁺ effect to eventually protect cells from lysis. The simultaneous recording of Na⁺ and K⁺ movements showed a significant increase of the K⁺ efflux although, the Na⁺ influx was reduced. By using potassium channels blockers, we found that, the potassium efflux enhanced by the presence of extracellular Ca²⁺, was markedly, inhibited in apamin-, charybdotoxin-, tetrodotoxin-, and quinine-pretreatment neutrophils. We also found that, the increase of the K⁺ efflux was reduced by either, thapsigargin or TMB8, potent blockers of the internal Ca²⁺ stores depletion. Consequently, we proposed that, the activation of another potassium pathway by leukotoxins, known as Ca²⁺-activated K⁺ channels following the Ca²⁺ stores depletion. Furthermore, potassium channels blockers did not affect ethidium, Na⁺ and K⁺ movements, in the absence of extracellular Ca²⁺. Moreover, in this condition, no monovalent ions movement was recorded, when the pores formation was altered by tetra-ethyl-ammonium. In the present study, we further highlighted the specificity of membrane pores to Na⁺ and K⁺ ions when, the pores formation was completely blocked by divalent ions blockers (Ca²⁺ and Zn²⁺). Under these conditions, no monovalent ions movement, was recorded although, a significant influx of Ca²⁺ and Zn²⁺ was observed after the leukotoxins application. In conclusion, our data provided an evidence that, staphylococcal leukotoxins induced in human neutrophils: 1) the opening of Ca²⁺-activated K⁺ channels, only in the presence of 1 mM extracellular Ca²⁺; 2) the formation of membrane pores, which exhibited a high specificity to monovalent cations and, 3) an influx of sodium, through a tetrodotoxin not-sensitive pathway ruling out the hypothesis that, Na⁺ channels could be activated by leukotoxins.

The Pore-forming Leukotoxins from <i>S. aureus</i> Involve Ca<sup>2+</sup> Release-Activated Ca<sup>2+</sup> Channels and Other Types of Ca<sup>2+</sup> Channels in Ca<sup>2+</sup> Entry into Neutrophils

The pore-forming bi-component leukotoxins from <i>Staphylococcus aureus</i> induce two independent cellular events <i>1)</i> the formation of trans-membrane pores not permeable to divalent ions and <i>2)</i> the opening of pre-existing Ca²⁺ channels in human polymorphonuclear neutrophils (PMNs). The influx of Ca²⁺ and Mn²⁺ (Mn²⁺ was used as a Ca²⁺ surrogate) in Fura2-loaded human PMNs was determined by spectrofluorometry techniques. The present study showed that, in the presence of extracellular Ca²⁺, the staphylococcal HlgA/HlgB γ-hemolysin induced a rapid Ca²⁺ release from internal Ca²⁺ stores before the onset of a Mn²⁺ (Ca²⁺) influx. The sustained increase of Ca²⁺ and Mn²⁺ influx was partially inhibited by the ionic blockers of Ca²⁺ Release-Activated Ca²⁺ (CRAC) channels, La³⁺ and Ni²⁺. Furthermore, the incubation of human PMNs with either TMB8 or thapsigargin did inhibit significantly the Ca²⁺ release mediated by leukotoxins simultanously to a clear decrease of Ca²⁺ and Mn²⁺ influx. The internal Ca²⁺ release induced by γ-hemolysin was also inhibited by PMNs pretreatment with a pertussis toxin, NaF, caffeine, ryanodine, cinnarizine and flunarizine and consequently, the Mn²⁺ (Ca²⁺) influx was significantly reduced. Moreover, different Ca²⁺ signaling pathways blockers such as U73122, staurosporine, thyrphostin A9 and okadaic acid were tested on the leukotoxins activity. Taken together, this work provided evidence that, in the presence of extracellular Ca²⁺, bi-component staphylococcal leukotoxins provoked in human PMNs after a specific binding to their membrane receptors, a rapid depletion of internal Ca²⁺ stores mediating a CRAC channels activation. This Ca²⁺-dependent mechanism seems likely to be associated to the heterotrimeric G-proteins activation. Interestingly, in the absence of extracellular Ca²⁺, the staphylococcal leukotoxins tested induced the opening of an important divalent ions (Ca²⁺, Mn²⁺, Ni²⁺) pathway not sensitive to CRAC channels blockers. Consequently, we strongly suggested that other types of Ca²⁺ channels might be involved in bi-component leukotoxins activity, including Ca²⁺ channels dependent on the protein kinase C activation.

Ethanol inhibition of NMDA receptors in calcium-dependent and –independent modes

N-methyl-d-aspartate receptor (NMDAR) is an essential target for ethanol action in the central nervous system (CNS). Whereas an alcohol addiction treatment represents a severe medical problem, many aspects of ethanol action at physiologically relevant concentrations on NMDARs are still unclear. Here using the whole-cell patch-clamp recording on cortical neurons in the primary culture, we studied inhibition of NMDAR currents by different ethanol concentrations ([Et]s) and its dependence on extracellular Ca2+. The ethanol action on NMDA-activated currents exhibited a biphasic concentration-inhibition relationship in the presence of extracellular Ca2+. The high-affinity region of the curve was found within the range of [Et]s from 9 mM to 30 mM and was characterized by IC50,H of about 20 mM. The low-affinity region was observed within the range of [Et]s from 85 mM to 200 mM with IC50,L of about 150 mM. In the absence of extracellular Ca2+, the ethanol concentration-inhibition relationship became monophasic, with IC50,L of about 200 mM, since the high-affinity component disappeared. A substitution of Li+ for Na+ in the bathing solution and an extraction of cholesterol from the plasma membrane with methyl-β-cyclodextrin, which are the treatments that both promote the Ca2+-dependent desensitization (CDD) of NMDARs, abolished the high-affinity Ca2+-dependent component of the NMDAR ethanol inhibition. Besides, this component was not observed when neurons were loaded with BAPTA. These data suggest that most likely, ethanol at low concentrations enhances the NMDAR CDD. In agreement when the dependence of the NMDAR CDD on extracellular Ca2+ was directly measured 22 mM ethanol enhanced the NMDAR CDD since an extracellular Ca2+ concentration that caused 50% of the NMDAR CDD decreased almost 3-folds from 0.81 mM to 0.28 mM, and an extent of the CDD was also more pronounced. The low-affinity component of the NMDAR ethanol inhibition was resistant to the above treatments suggesting CDD-independent direct action on NMDARs.Thus, at a physiologically relevant concentration of extracellular Ca2+ and ethanol that could be reached in the blood during light-mild human alcohol intoxication, ethanol causes an enhancement of the NMDAR CDD, which could be in general accompanied by some disruptions of the CNS excitatory system.

Tonic Calcium-Activated Chloride Current Sustained by ATP Release and Highly Desensitizing Human P2X1 Receptors

Extracellular adenosine triphosphate (ATP) participates in maintaining the vascular tone in the CNS, particularly in the retina, via the tonic activity of ligand gated activated P2X1 receptors. P2X1 receptors are characterized by their high affinity for ATP and their strong desensitization to concentrations of ATP that are 200-fold lower than their EC50. The mechanism behind P2X1 tonic activity remains unclear. In this study, we expressed human P2X1 (hP2X1) homomeric receptors in Xenopus oocytes to explore the relationship between ATP release from oocytes at rest, hP2X1, and Ca2+-activated Cl− channels. Our results indicate that Xenopus oocytes release ATP at rest via vesicular exocytosis, and this process is a constitutive phenomenon independent of extracellular Ca2+. Our results also indicate that hP2X1 receptors are able to sustain a tonic activity of Ca2+-activated Cl− channels. In the presence of extracellular Ca2+ the activity of hP2X1 receptors is greatly amplified by its coupling with Ca2+-activated Cl− channels. Future studies addressing the relationship between hP2X1 receptors and Ca2+-activated Cl− channels in vascular smooth muscle cells should provide information about additional mechanisms that regulate the vascular tone and their potential as pharmaceutical targets. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries.

Long-term dexamethasone treatment diminishes store-operated Ca2+ entry in salivary acinar cells

Corticosteroids are used in the treatment of many diseases; however, they also induce various side effects. Dexamethasone is one of the most potent corticosteroids, and it has been reported to induce the side effect of impaired salivary gland function. This study aimed to evaluate the effects of dexamethasone on mouse submandibular gland function to gain insight into the mechanism of dexamethasone-induced salivary hypofunction. The muscarinic agonist carbachol (CCh) induced salivary secretion and was not affected by short-term dexamethasone treatment but was decreased following long-term dexamethasone administration. The expression levels of the membrane proteins Na+-K+-2Cl− cotransporter, transmembrane member 16A, and aquaporin 5 were comparable between the control and long-term dexamethasone treatment groups. The CCh-induced increase in calcium concentration was significantly lower in the presence of extracellular Ca2+ in the long-term dexamethasone treatment group compared to that in the control group. Furthermore, CCh-induced salivation in the absence of extracellular Ca2+ and Ca2+ ionophore A23187-induced salivation was comparable between the control and long-term dexamethasone treatment groups. Moreover, salivation induced by the Ca2+-ATPase inhibitor thapsigargin was diminished in the long-term dexamethasone treatment group. In summary, these results demonstrate that short-term dexamethasone treatment did not impair salivary gland function, whereas long-term dexamethasone treatment diminished store-operated Ca2+ entry, resulting in hyposalivation in mouse submandibular glands.

Variations of Intracellular Ca2+ Mobilization Initiated by Nanosecond and Microsecond Electrical Pulses in HeLa Cells.

Herein, the variations in transient Ca2+ mobilizations in HeLa cells exposed to a single, non-thermal pulsed electric field (PEF) are described. Three PEF waveforms categorized by pulse duration and intensity were used to deduce the kinetics involved in Ca2+ mobilization. A fast microscopic fluorescent imaging system and a fluorescent molecular probe were used to observe transient intracellular Ca2+ mobilization after pulse exposure. The sources and pathways in the transient Ca2+ mobilizations were investigated using an inhibitor of inositol-1,4,5-trisphosphate receptor (IP3R) on the endoplasmic reticulum (ER) along with a Ca2+-free buffer. When exposed to the 10-μs-long PEF, the Ca2+ concentration increased mainly at the cathodic region near the membrane. However, Ca2+ concentration increased at both anodic and cathodic regions when Na+ concentration in the buffer was reduced. Ca2+ concentration increased only in the presence of extracellular Ca2+. These results suggest that the 10-μs PEF takes a large amount of extracellular Na+ into the cell through the electropermeabilized plasma membrane, especially at the anodic side, resulting in the suppression of the Ca2+ influx. On the contrary, the 20-ns-long PEF increased Ca2+ concentration in the surrounding region of the nucleus only in the presence of extracellular Ca2+. The PEF exposure with inhibition of the IP3R indicates that increased Ca2+ ions are released from the ER via the activated IP3R. These mechanisms could induce specific cell responses, such as Ca2+ oscillations, Ca2+ waves, and Ca2+ puffs.

Sensing of L-Arginine by Gut-Expressed Calcium Sensing Receptor Stimulates Gut Satiety Hormones Cholecystokinin and Glucose-Dependent Insulinotropic Peptide Secretion in Pig Model.

Nutrients regulate the secretion of gut satiety hormones, which is related to the modulation of food intake and blood glucose levels. Calcium-sensing receptor (CaSR) is involved in regulating gut hormone secretion in response to l-amino acids and multivalent cations. Rodents are often used to investigate the effect of nutrients on these hormonal release. However, results obtained using rodent models are difficult to be applied in humans, we used pigs as a model in this study because their physiology is similar to that of humans. In this study, we investigated whether l-Arginine (l-Arg) could induce gut hormones cholecystokinin (CCK) and glucose-dependent insulinotropic peptide (GIP) secretion in the porcine duodenum and if so, whether CaSR mediated l-Arg-regulated gut satiety hormone secretion. Our data showed that treatment with 20 and 50 mM l-Arg induced CCK and GIP secretion compared with 0 mM l-Arg. However, treatment with d-Arg (an inactive isomer) failed to elicit this response. The potency of l-Arg to induce CCK and GIP secretion was enhanced in the presence of extracellular Ca2+ and CaSR agonist cinacalcet. However, the effect of Arg on CCK and GIP secretion was attenuated by blocking CaSR and its downstream signaling molecules adenylate cyclase (AC) and phospholipase C (PLC). Taken all together, pig duodenum provides an appropriate model to explore the effects of l-Arg on the secretion of the satiety-related gut hormones CCK and GIP and the role of CaSR in this effect. Further investigations are needed to verify the effect of l-Arg on food intake and blood glucose in human study. PRACTICAL APPLICATION: l-Arginine is able to modulate cholecystokinin and glucose-dependent insulinotropic peptide secretion through the CaSR in pig model, which has a potential role in regulating food intake and blood glucose levels.