Removal Of Extracellular Ca2 Research Articles

Substance P Activates Ca2+-Permeable Nonselective Cation Channels through a Phosphatidylcholine-Specific Phospholipase C Signaling Pathway in nNOS-Expressing GABAergic Neurons in Visual Cortex.

To understand the functions of the neocortex, it is essential to characterize the properties of neurons constituting cortical circuits. Here, we focused on a distinct group of GABAergic neurons that are defined by a specific colocalization of intense labeling for both neuronal nitric oxide synthase (nNOS) and substance P (SP) receptor [neurokinin 1 (NK1) receptors]. We investigated the mechanisms of the SP actions on these neurons in visual cortical slices obtained from young glutamate decarboxylase 67-green fluorescent protein knock-in mice. Bath application of SP induced a nonselective cation current leading to depolarization that was inhibited by the NK1 antagonists in nNOS-immunopositive neurons. Ruthenium red and La(3+), transient receptor potential (TRP) channel blockers, suppressed the SP-induced current. The SP-induced current was mediated by G proteins and suppressed by D609, an inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), but not by inhibitors of phosphatidylinositol-specific PLC, adenylate cyclase or Src tyrosine kinases. Ca(2+) imaging experiments under voltage clamp showed that SP induced a rise in intracellular Ca(2+) that was abolished by removal of extracellular Ca(2+) but not by depletion of intracellular Ca(2+) stores. These results suggest that SP regulates nNOS neurons by activating TRP-like Ca(2+)-permeable nonselective cation channels through a PC-PLC-dependent signaling pathway.

Stimulation of suicidal erythrocyte death by sulforaphane.

Sulforaphane, an isothiocyanate from cruciferous vegetable, counteracts malignancy. The effect is at least in part due to the stimulation of suicidal death or apoptosis of tumour cells. Mechanisms invoked in sulforaphane-induced apoptosis include mitochondrial depolarization and altered gene expression. Despite the lack of mitochondria and nuclei, erythrocytes may, similar to apoptosis of nucleated cells, enter eryptosis, a suicidal cell death characterized by cell shrinkage and phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)]i). This study explored whether sulforaphane stimulates eryptosis. Cell volume was estimated from forward scatter, phosphatidylserine exposure at the cell surface from annexin V binding and [Ca(2+)]i from Fluo-3 fluorescence. A 48-hr treatment of human erythrocytes with sulforaphane (50-100 μM) significantly decreased forward scatter, significantly increased the percentage of annexin V binding cells and significantly increased [Ca(2+)]i. The effect of sulforaphane (100 μM) on annexin V binding was significantly blunted but not abrogated by the removal of extracellular Ca(2+). Sulforaphane (100 μM) significantly increased ceramide formation. In conclusion, sulforaphane stimulates suicidal erythrocyte death or eryptosis, an effect at least partially, but not exclusively, due to the stimulation of Ca(2+) entry and ceramide formation.

In vitro sensitization of erythrocytes to programmed cell death following baicalein treatment.

The polyphenolic flavonoid Baicalein has been shown to trigger suicidal death or apoptosis of tumor cells and is thus considered for the prevention and treatment of malignancy. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and ceramide. The present study explored whether Baicalein stimulates eryptosis. To this end, forward scatter was taken for measurement of cell volume, annexin-V-binding for phosphatidylserine-exposure, Fluo3 fluorescence for [Ca2+]i and fluorescent antibodies for ceramide abundance. As a result, a 48 h exposure of human erythrocytes to Baicalein was followed by significant decrease of forward scatter (≥10 µM), significant increase of the percentage of annexin-V-binding cells (≥25 µM), significant increase of [Ca2+]i (50 µM) and significant increase of ceramide abundance (50 µM). The effect of Baicalein (50 µM) on annexin-V-binding was significantly blunted but not abrogated by removal of extracellular Ca2+. In conclusion, at the concentrations employed, Baicalein stimulates suicidal erythrocyte death or eryptosis, an effect at least in part due to the combined effects of Ca2+ entry and ceramide formation.

P2Y13 receptor-mediated rapid increase in intracellular calcium induced by ADP in cultured dorsal spinal cord microglia.

P2Y receptors have been implicated in the calcium mobilization by the response to neuroexcitatory substances in neurons and astrocytes, but little is known about P2Y receptors in microglia cells. In the present study, the effects of ADP on the intracellular calcium concentration ([Ca(2+)]i) in cultured dorsal spinal cord microglia were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescence indicator that could monitor real-time alterations of [Ca(2+)]i. Here we show that ADP (0.01-100 μM) causes a rapid increase in [Ca(2+)]i with a dose-dependent manner in cultured microglia. The action of ADP on [Ca(2+)]i was significantly blocked by MRS2211 (a selective P2Y13 receptor antagonist), but was unaffected by MRS2179 (a selective P2Y1 receptor antagonist) or MRS2395 (a selective P2Y12 receptor antagonist), which suggest that P2Y13 receptor may be responsible for ADP-evoked Ca(2+) mobilization in cultured microglia. P2Y13-evoked Ca(2+) response can be obviously inhibited by BAPTA-AM and U-73122, respectively. Moreover, removal of extracellular Ca(2+) (by EGTA) also can obvious suppress the Ca(2+) mobilization. These results means both intracellular calcium and extracellular calcium are potentially important mechanisms in P2Y13 receptor-evoked Ca(2+) mobilization. However, P2Y13 receptor-evoked Ca(2+) response was not impaired after CdCl2 and verapamil administration, which suggest that voltage-operated Ca(2+) channels may be not related with P2Y13-evoked Ca(2+) response. In addition, Ca(2+) mobilization induced by ADP was abolished by different store-operated Ca(2+) channels (SOCs) blocker, 2-APB (50 μM) and SKF-96365 (1 mM), respectively. These observations suggest that the activation of P2Y13 receptor might be involved in the effect of ADP on [Ca(2+)]i in cultured dorsal spinal cord microglia. Furthermore, our results raise a possibility that P2Y13 receptor activation causes Ca(2+) release from Ca(2+) store, which leads to the opening of SOCs.

The mechanism of honokiol-induced intracellular Ca2+ rises and apoptosis in human glioblastoma cells

Honokiol, an active constituent of oriental medicinal herb Magnolia officinalis, caused Ca2+ mobilization and apoptosis in different cancer cells. In vivo, honokiol crossed the blood–brain or –cerebrospinal fluid barrier, suggesting that it may be an effective drug for the treatment of brain tumors, including glioblastoma. This study examined the effect of honokiol on intracellular Ca2+ concentration ([Ca2+]i) and apoptosis in DBTRG-05MG human glioblastoma cells. Honokiol concentration-dependently induced a [Ca2+]i rise. The signal was decreased partially by removal of extracellular Ca2+. Honokiol-triggered [Ca2+]i rise was not suppressed by store-operated Ca2+ channel blockers (nifedipine, econazole, SK&F96365) and the protein kinase C (PKC) activator phorbol 12-myristate 13 acetate (PMA), but was inhibited by the PKC inhibitor GF109203X. GF109203X-induced inhibition was not altered by removal of extracellular Ca2+. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished honokiol-induced [Ca2+]i rise. Conversely, incubation with honokiol abolished TG or BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C (PLC) with U73122 abolished honokiol-induced [Ca2+]i rise. Honokiol (20–80μM) reduced the cell viability, which was not reversed by prechelating cytosolic Ca2+ with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester). Honokiol (20–60μM) enhanced reactive oxygen species (ROS) production, decreased mitochondrial membrane potential, released cytochrome c, and activated caspase-9/caspase-3. Together, honokiol induced a [Ca2+]i rise by inducing PLC-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via PKC-dependent, non store-operated Ca2+ channels. Moreover, honokiol activated the mitochondrial pathway of apoptosis in DBTRG-05MG human glioblastoma cells.

Sensitization of cardiac Ca²⁺ release sites by protein kinase C signaling: evidence from action of murrayafoline A.

In the present study, we explored the effects of a plant alkaloid compound, 1-methoxy-3-methylcarbazole (murrayafoline A, Mu-A), on focal and global Ca(2+) signaling, and the underlying cellular mechanisms. Rapid two-dimensional confocal Ca(2+) imaging and image analysis were used to measure Ca(2+) signals in rat ventricular myocytes. Application of Mu-A (10-100 μM) significantly enhanced the magnitude and rate of Ca(2+) release on depolarization with no change in Ca(2+) transient decay. Focal Ca(2+) release events (Ca(2+) sparks) occurred more often, and their duration and size were greater after the application of Mu-A. In addition, sarcoplasmic reticulum (SR) Ca(2+) loading and fractional release were increased by exposure to Mu-A. All these effects reached steady state within 2-3 min after Mu-A application. The higher occurrence of Ca(2+) sparks in the presence of Mu-A was resistant to SR Ca(2+) clamping, removal of extracellular Ca(2+) and Na(+), and blockade of either protein kinase A, Ca(2+)/calmodulin-dependent protein kinase II, phospholipase C, or inositol 1,4,5-trisphosphate receptors, but it was abolished by the inhibition of protein kinase C (PKC). SR Ca(2+) clamping prevented the Mu-A-induced Ca(2+) spark prolongation and enlargement. The Mu-A-induced enhancement of Ca(2+) transients was also eliminated by PKC blockade. Mu-A enhanced PKC activity in vitro. These results suggest that Mu-A may increase spark occurrence via its direct enhancement of PKC activity and subsequent sensitization of ryanodine receptor clusters and that this mechanism, as well as increased SR Ca(2+) loading, may partly explain larger and more rapid global Ca(2+) releases in the presence of Mu-A during depolarization.

Extracellular Ca2+per se inhibits quantal size of catecholamine release in adrenal slice chromaffin cells

Classic calcium hypothesis states that depolarization-induced increase in intracellular Ca2+ concentration ([Ca2+]i) triggers vesicle exocytosis by increasing vesicle release probability in neurons and neuroendocrine cells. The extracellular Ca2+, in this calcium hypothesis, serves as a reservoir of Ca2+ source. Recently we find that extracellular Ca2+per se inhibits the [Ca2+]i dependent vesicle exocytosis, but it remains unclear whether quantal size is regulated by extracellular, or intracellular Ca2+ or both [1]. In this work we showed that, in physiological condition, extracellular Ca2+per se specifically inhibited the quantal size of single vesicle release in rat adrenal slice chromaffin cells. The extracellular Ca2+ in physiological concentration (2.5mM) directly regulated fusion pore kinetics of spontaneous quantal release of catecholamine. In addition, removal of extracellular Ca2+ directly triggered vesicle exocytosis without eliciting intracellular Ca2+. We propose that intracellular Ca2+ and extracellular Ca2+per se cooperately regulate single vesicle exocytosis. The vesicle release probability was jointly modulated by both intracellular and extracellular Ca2+, while the vesicle quantal size was mainly determined by extracellular Ca2+ in chromaffin cells physiologically.

Ca2+ Influx via the Na+/Ca2+ Exchanger Is Enhanced in Malignant Hyperthermia Skeletal Muscle

Malignant hyperthermia (MH) is potentially fatal pharmacogenetic disorder of skeletal muscle caused by intracellular Ca(2+) dysregulation. NCX is a bidirectional transporter that effluxes (forward mode) or influxes (reverse mode) Ca(2+) depending on cellular activity. Resting intracellular calcium ([Ca(2+)]r) and sodium ([Na(+)]r) concentrations are elevated in MH susceptible (MHS) swine and murine muscles compared with their normal (MHN) counterparts, although the contribution of NCX is unclear. Lowering [Na(+)]e elevates [Ca(2+)]r in both MHN and MHS swine muscle fibers and it is prevented by removal of extracellular Ca(2+) or reduced by t-tubule disruption, in both genotypes. KB-R7943, a nonselective NCX3 blocker, reduced [Ca(2+)]r in both swine and murine MHN and MHS muscle fibers at rest and decreased the magnitude of the elevation of [Ca(2+)]r observed in MHS fibers after exposure to halothane. YM-244769, a high affinity reverse mode NCX3 blocker, reduces [Ca(2+)]r in MHS muscle fibers and decreases the amplitude of [Ca(2+)]r rise triggered by halothane, but had no effect on [Ca(2+)]r in MHN muscle. In addition, YM-244769 reduced the peak and area under the curve of the Ca(2+) transient elicited by high [K(+)]e and increased its rate of decay in MHS muscle fibers. siRNA knockdown of NCX3 in MHS myotubes reduced [Ca(2+)]r and the Ca(2+) transient area induced by high [K(+)]e. These results demonstrate a functional NCX3 in skeletal muscle whose activity is enhanced in MHS. Moreover reverse mode NCX3 contributes to the Ca(2+) transients associated with K(+)-induced depolarization and the halothane-triggered MH episode in MHS muscle fibers.

Arachidonate-Regulated Ca2+ Influx in Human Airway Smooth Muscle

Plasma membrane Ca(2+) influx, especially store-operated Ca(2+) entry triggered by sarcoplasmic reticulum (SR) Ca(2+) release, is a key component of intracellular calcium concentration ([Ca(2+)]i) regulation in airway smooth muscle (ASM). Agonist-induced Ca(2+) oscillations in ASM that involve both influx and SR mechanisms have been previously demonstrated. In nonexcitable cells, [Ca(2+)]i oscillations involve Ca(2+) influx via arachidonic acid (AA) -stimulated channels, which show similarities to store-operated Ca(2+) entry, although their molecular identity remains undetermined. Little is known about AA-regulated Ca(2+) channels or their regulation in ASM. In enzymatically dissociated human ASM cells loaded with the Ca(2+) indicator, fura-2, AA (1-10 μM) triggered [Ca(2+)]i oscillations that were inhibited by removal of extracellular Ca(2+). Other fatty acids, such as the diacylglycerol analog, 1-oleoyl-2-acetyl-SN-glycerol, oleic acid, and palmitic acid (10 μM each), failed to elicit similar [Ca(2+)]i responses. Preincubation with LaCl3 (1 μM or 1 mM) inhibited AA-induced oscillations. Inhibition of receptor-operated channels (SKF96,365 [10 μM]), lipoxygenase (zileuton [10 μM]), or cyclooxygenase (indomethacin [10 μM]) did not affect oscillation parameters. Inhibition of SR Ca(2+) release (ryanodine [10 μM] or inositol 1,4,5-trisphosphate receptor inhibitor, xestospongin C [1 μM]) decreased [Ca(2+)]i oscillation frequency and amplitude. Small interfering RNA against caveolin-1, stromal interaction molecule 1, or Orai3 (20 nM each) reduced the frequency and amplitude of AA-induced [Ca(2+)]i oscillations. In ASM cells derived from individuals with asthma, AA increased oscillation amplitude, but not frequency. These results are highly suggestive of a novel AA-mediated Ca(2+)-regulatory mechanism in human ASM, reminiscent of agonist-induced oscillations. Given the role of AA in ASM intracellular signaling, especially with inflammation, AA-regulated Ca(2+) channels could potentially contribute to increased [Ca(2+)]i in diseases such asthma.

Suppression of cell membrane permeability by suramin: involvement of its inhibitory actions on connexin 43 hemichannels

Suramin is a clinically prescribed drug for treatment of human African trypanosomiasis, cancer and infection. It is also a well-known pharmacological antagonist of P2 purinoceptors. Despite its clinical use and use in research, the biological actions of this molecule are still incompletely understood. Here, we investigated the effects of suramin on membrane channels, as exemplified by its actions on non-junctional connexin43 (Cx43) hemichannels, pore-forming α-haemolysin and channels involved in ATP release under hypotonic conditions. Hemichannels were activated by removing extracellular Ca(2+) . The influences of suramin on hemichannel activities were evaluated by its effects on influx of fluorescent dyes and efflux of ATP. The membrane permeability and integrity were assessed through cellular retention of preloaded calcein and LDH release. Suramin blocked Cx43 hemichannel permeability induced by removal of extracellular Ca(2+) without much effect on Cx43 expression and gap junctional intercellular communication. This action of suramin was mimicked by its analogue NF023 and NF449 but not by another P2 purinoceptor antagonist PPADS. Besides hemichannels, suramin also significantly blocked intracellular and extracellular exchanges of small molecules caused by α-haemolysin from Staphylococcus aureus and by exposure of cells to hypotonic solution. Furthermore, it prevented α-haemolysin- and hypotonic stress-elicited cell injury. Suramin blocked membrane channels and protected cells against toxin- and hypotonic stress-elicited injury. Our finding provides novel mechanistic insights into the pharmacological actions of suramin. Suramin might be therapeutically exploited to protect membrane integrity under certain pathological situations.

In vitro induction of erythrocyte phosphatidylserine translocation by the natural naphthoquinone shikonin.

Shikonin, the most important component of Lithospermum erythrorhizon, has previously been shown to exert antioxidant, anti-inflammatory, antithrombotic, antiviral, antimicrobial and anticancer effects. The anticancer effect has been attributed to the stimulation of suicidal cell death or apoptosis. Similar to the apoptosis of nucleated cells, erythrocytes may experience eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include the increase of cytosolic Ca2+-activity ([Ca2+]i) and ceramide formation. The present study explored whether Shikonin stimulates eryptosis. To this end, Fluo 3 fluorescence was measured to quantify [Ca2+]i, forward scatter to estimate cell volume, annexin V binding to identify phosphatidylserine-exposing erythrocytes, hemoglobin release to determine hemolysis and antibodies to quantify ceramide abundance. As a result, a 48 h exposure of human erythrocytes to Shikonin (1 µM) significantly increased [Ca2+]i, increased ceramide abundance, decreased forward scatter and increased annexin V binding. The effect of Shikonin (1 µM) on annexin V binding was significantly blunted, but not abolished by the removal of extracellular Ca2+. In conclusion, Shikonin stimulates suicidal erythrocyte death or eryptosis, an effect at least partially due to the stimulation of Ca2+ entry and ceramide formation.

Effects of Thymol on Ca^(2+) Homeostasis and Apoptosis in MDCK Renal Tubular Cells

Thymol is a natural essential oil present in many plants and has many different effects in various cell types. However, the effect of thymol on the physiology of MDCK renal tubular cells is unknown. The action of the phytochemical thymol on cytosolic Ca²⁺ concentrations ([Ca²⁺]i) and apoptosis in Madin-Darby canine kidney (MDCK) renal tubular cells was explored. Fura-2, a Ca²⁺-sensitive fluorescent dye, was used to assess [Ca²⁺]i. Thymol at concentrations of 200-500 μM caused a [Ca²⁺]i rise in a concentration-dependent manner. Removal of extracellular Ca²⁺ partially reduced the effects of thymol. Thymol-induced Ca²⁺ entry was inhibited by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In a Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin inhibited thymol-induced [Ca²⁺]i increases. Treatment with thymol also inhibited thapsigargin-induced [Ca²⁺]i rise. Thymol killed cells at concentrations of 300-500 μM in a concentrationdependent fashion. Chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not prevent thymol cytotoxicity. Thymol (400 and 500 μM) induced apoptosis detected by using Annexin V/propidium iodide staining. At 400 or 500 μM, thymol increased levels of reactive oxygen species. Together, in MDCK cells, thymol induced a [Ca²⁺]i rise by inducing Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-sensitive store-operated Ca²⁺ channels. Our data suggest that thymol-induced apoptosis might involve reactive oxygen species (ROS) production.

L-type Ca2+ channel sparklets revealed by TIRF microscopy in mouse urinary bladder smooth muscle.

Calcium is a ubiquitous second messenger in urinary bladder smooth muscle (UBSM). In this study, small discrete elevations of intracellular Ca2+, referred to as Ca2+ sparklets have been detected in an intact detrusor smooth muscle electrical syncytium using a TIRF microscopy Ca2+ imaging approach. Sparklets were virtually abolished by the removal of extracellular Ca2+ (0.035±0.01 vs. 0.23±0.07 Hz/mm2; P<0.05). Co-loading of smooth muscle strips with the slow Ca2+ chelator EGTA-AM (10 mM) confirmed that Ca2+ sparklets are restricted to the cell membrane. Ca2+ sparklets were inhibited by the calcium channel inhibitors R-(+)-Bay K 8644 (1 μM) (0.034±0.02 vs. 0.21±0.08 Hz/mm2; P<0.05), and diltiazem (10 μM) (0.097±0.04 vs. 0.16±0.06 Hz/mm2; P<0.05). Ca2+ sparklets were unaffected by inhibition of P2X1 receptors α,β-meATP (10 μM) whilst sparklet frequencies were significantly reduced by atropine (1 μM). Ca2+ sparklet frequency was significantly reduced by PKC inhibition with Gö6976 (100 nM) (0.030±0.01 vs. 0.30±0.1 Hz/mm2; P<0.05), demonstrating that Ca2+ sparklets are PKC dependant. In the presence of CPA (10 μM), there was no apparent change in the overall frequency of Ca2+ sparklets, although the sparklet frequencies of each UBSM became statistically independent of each other (Spearman's rank correlation 0.2, P>0.05), implying that Ca2+ store mediated signals regulate Ca2+ sparklets. Under control conditions, inhibition of store operated Ca2+ entry using ML-9 (100 μM) had no significant effect. Amplitudes of Ca2+ sparklets were unaffected by any agonists or antagonists, suggesting that these signals are quantal events arising from activation of a single channel, or complex of channels. The effects of CPA and ML-9 suggest that Ca2+ sparklets regulate events in the cell membrane, and contribute to cytosolic and sarcoplasmic Ca2+ concentrations.

Distinct mechanisms of [Ca2+]i responses to flow and hypotonicity in the distal nephron (892.5)

Changes in luminal fluid flow or osmolality elicit [Ca2+]i elevations in the distal nephron (DN) cells to regulate water‐electrolyte transport. Our group has recently identified a pivotal role of TRPV4 in mediating flow‐induced Ca2+ elevations in the DN cells, however little is known about molecular nature of the response to osmotic gradients. Here, using ratiometric Fura‐2 Ca2+ imaging in freshly isolated split‐opened DNs from wild‐type (WT) mice, we found that hypotonicity elicits a uniform and reproducible increase of [Ca2+]i in DN cells. This response is abolished by removal of extracellular Ca2+, but only moderately diminished upon depletion of intracellular Ca2+ stores. While genetic ablation of TRPV4 abrogates flow‐induced [Ca2+]i elevations, the response to hypotonicity, though significantly blunted, is retained in TRPV4‐/‐ mice. This points to additional mechanisms increasing [Ca2+]i in response to osmotic gradients. Indeed, pharmacological inhibition of Ca2+‐permeable TRPC3 channel markedly diminishes the amplitude of [Ca2+]i elevations induced by hypotonic stimuli in DN epithelium from WT mice. Moreover, the threshold of the response to decreased osmolality is elevated in TRPC3 knockouts. Thus, in contrast to flow‐induced [Ca2+]i elevations mediated by TRPV4, hypotonicity elicits a multifaceted Ca2+ response, at least in part due to coordinated activation of TRPV4 and TRPC3.Grant Funding Source: DK095029

Extracellular ADP‐ribose induces [Ca2+]i increase via purinergic pathway in pulmonary artery smooth muscle cells (1175.4)

ADP‐ribose (ADPR) is the product of NAD metabolism and cyclic ADPR hydrolysis by CD38, and poly(ADPR) degradation by poly(ADPR) glycohydrolase. It is recognized as a signaling molecule for the activation of the transient receptor potential melastatin 2 (TRPM2) channel and play important roles in H2O2 induced response. In the plasma membrane, CD38 catalytic site orients both extra‐ and intracellularly. Hence, ADPR can be generated and release outside of the cells. However, the role of extracellular ADPR is unclear. Recent studies suggest that ADPR can activate purinergic receptors in pancreaticβcells. Here we examined the Ca2+ mobilizing effect of extracellular ADPR in rat pulmonary artery smooth muscle cells (PASMCs). Application of ADPR (30 µM to 1 mM) elicited concentration dependent increase in [Ca2+]i of PASMCs. At 1 mM, ADPR induced Ca2+ response with a transient and a sustained phase. The sustained phase was abolished by the removal of extracellular Ca2+. Suramin (a non‐selective P2 receptor antagonist) attenuated ADPR‐induced sustain phase, and PPADS (a P2X receptor antagonist) inhibited the peak response. MRS2179 (a selective P2Y1 receptor inhibitor) had no effect on ADPR‐induced Ca2+ mobilization. The PLC inhibitor U73122, but not its inactive analog U73343, inhibited the ADPR‐induced response by 70%. U73122 plus PPADS further decreased the ADPR‐induced peak response. 2‐APB inhibited ADPR‐induced peak response but not the sustain phase. These data indicate that ADPR is an agonist of purinergic receptors, and it mobilizes [Ca2+]i via P2 receptor‐PLC‐IP3R calcium release pathway, but not through TRPM2‐mediated Ca2+ entry. (Supported by R01 HL071835 and R01 HL075134)

Molecular mechanism of 2-APB-induced Ca2+ influx in external acidification in PC12

2-Aminoethoxydiphenyl borate (2-APB) is used as a pharmacological tool because it antagonizes inositol 1,4,5-trisphosphate receptors and store-operated Ca2+ (SOC) channels, and activates some TRP channels. Recently, we reported that 2-APB enhanced the increase in cytotoxic [Ca2+]i, resulting in cell death under external acidic conditions in rat pheochromocytoma cell line PC12. However, the molecular mechanism and functional role of the 2-APB-induced Ca2+ influx in PC12 have not been clarified. In this study, to identify the possible target for the action of 2-APB we examined the pharmacological and molecular properties of [Ca2+]i and secretory responses to 2-APB under extracellular low pH conditions. 2-APB dose-dependently induced a [Ca2+]i increase and dopamine release, which were greatly enhanced by the external acidification (pH 6.5). [Ca2+]i and secretory responses to 2-APB at pH 6.5 were inhibited by the removal of extracellular Ca2+ and SOC channel blockers such as SK&F96365, La3+ and Gd3+. PC12 expressed all SOC channel molecules, Orai 1, Orai 2 and Orai 3. When we used an siRNA system, downregulation of Orai 3, but not Orai 1 and Orai 2, attenuated both [Ca2+]i and secretory responses to 2-APB. These results suggest that 2-APB evokes external acid-dependent increases of [Ca2+]i and dopamine release in PC12 through the activation of Orai 3. The present results indicate that 2-APB may be a useful pharmacological tool for Orai channel-related signaling.

Implication of transient receptor potential vanilloid type 1 in 14,15-epoxyeicosatrienoic acid-induced angiogenesis.

14,15-epoxyeicosatrienoic acid (14,15-EET) is implicated in regulating physiological functions of endothelial cells (ECs), yet the potential molecular mechanisms underlying the beneficial effects in ECs are not fully understood. In this study, we investigated whether transient receptor potential vanilloid receptor type 1 (TRPV1) is involved in 14,15-EET-mediated Ca2+ influx, nitric oxide (NO) production and angiogenesis. In human microvascular endothelial cells (HMECs), 14,15-EET time-dependently increased the intracellular level of Ca2+. Removal of extracellular Ca2+, pharmacological inhibition or genetic disruption of TRPV1 abrogated 14,15-EET-mediated increase of intracellular Ca2+ level in HMECs or TRPV1-transfected HEK293 cells. Furthermore, removal of extracellular Ca2+ or pharmacological inhibition of TRPV1 decreased 14,15-EET-induced NO production. 14,15-EET-mediated tube formation was abolished by TRPV1 pharmacological inhibition. In an animal experiment, 14,15-EET-induced angiogenesis was diminished by inhibition of TRPV1 and in TRPV1-deficient mice. TRPV1 may play a crucial role in 14,15-EET-induced Ca2+ influx, NO production and angiogenesis.

Stimulation of Erythrocyte Cell Membrane Scrambling by Gedunin

Background/Aims: Gedunin, an inhibitor of heat shock protein HSP90, triggers apoptosis of tumor cells and is thus effective against malignancy. Moreover, the drug has antimalarial potency. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and by phosphatidylserine translocation to the erythrocyte surface. Eryptosis may be triggered by increase of cytosolic Ca²⁺-activity ([Ca²⁺]_i). The present study explored whether gedunin stimulates eryptosis. Methods: Forward scatter was determined to estimate cell volume, annexin V binding to identify phosphatidylserine-exposing erythrocytes, hemoglobin release to depict hemolysis, and Fluo3-fluorescence to quantify [Ca²⁺]_i. Results: A 48 h exposure of human erythrocytes to gedunin significantly increased [Ca²⁺]_i (12 µM), significantly decreased forward scatter (24 µM) and significantly increased annexin-V-binding (12 µM). The effect of gedunin (24 µM) on annexin-V-binding was virtually abrogated by removal of extracellular Ca²⁺. Conclusion: Gedunin stimulates suicidal erythrocyte death or eryptosis, an effect mainly if not exclusively due to stimulation of Ca²⁺ entry.

Ca2+ Influx Mediated by Reverse Mode of Na+/Ca2+ Exchanger is Enhanced in Malignant Hyperthermia Skeletal Muscle

Malignant hyperthermia (MH), is potentially fatal pharmacogenetic disorder of skeletal muscle, which has been associated to intracellular Ca2+ dysregulation. The Na+/Ca2+ exchange (NCX) is a bidirectional transporter that normally extrudes Ca2+ from the cell (forward mode), but also brings Ca2+ into the cell (reverse mode) under special conditions such as intracellular Na+ accumulation or membrane depolarization. Resting intracellular Ca2+ concentration ([Ca2+]r) and intracellular Na+ concentration ([Na+]r) were elevated in MH susceptible (MHS) swine and mouse muscles compared to Wt (MHN) muscle fibers. Lowering extracellular Na+ induced an elevation of [Ca2+]r in both MHN and MHS swine and rodent muscle fibers that could be prevented by removal of extracellular Ca2+. Local application of KB-R7943, a nonselective NCX blocker, reduced [Ca2+]r in both MHN and MHS muscle fibers and the magnitude of the elevation of [Ca2+]r observed during a MH episode. YM-2444769 an NCX blocker that preferentially inhibits NCX3 reverse mode did not reduce resting [Ca2+]r in MHN, but it does in MHS muscle fibers, and decreased the amplitude of the elevation [Ca2+]r induced by halothane in these muscles. These results demonstrate the existence of a functional NCX in skeletal muscle, which appears to be enhanced in MHS muscle fibers. In addition, they represent the first evidence that NCX -reverse mode- contributes to the dysfunction of [Ca2+]r and [Na+]r and intracellular Ca2+ elevation induced by halothane in MHS muscle.Funding: NIH AR43140, AR052534 (PDA, JRL)

Cyanidin-3-glucoside Inhibits ATP-induced Intracellular Free Ca(2+) Concentration, ROS Formation and Mitochondrial Depolarization in PC12 Cells.

Flavonoids have an ability to suppress various ion channels. We determined whether one of flavonoids, cyanidin-3-glucoside, affects adenosine 5'-triphosphate (ATP)-induced calcium signaling using digital imaging methods for intracellular free Ca2+ concentration ([Ca2+]i), reactive oxygen species (ROS) and mitochondrial membrane potential in PC12 cells. Treatment with ATP (100µM) for 90 sec induced [Ca2+]i increases in PC12 cells. Pretreatment with cyanidin-3-glucoside (1µ g/ml to 100µg/ml) for 30 min inhibited the ATP-induced [Ca2+]i increases in a concentration-dependent manner (IC50=15.3µg/ml). Pretreatment with cyanidin-3-glucoside (15µg/ml) for 30 min significantly inhibited the ATP-induced [Ca2+]i responses following removal of extracellular Ca2+ or depletion of intracellular [Ca2+]i stores. Cyanidin-3-glucoside also significantly inhibited the relatively specific P2X2 receptor agonist 2-MeSATP-induced [Ca2+]i responses. Cyanidin-3-glucoside significantly inhibited the thapsigargin or ATP-induced store-operated calcium entry. Cyanidin-3-glucoside significantly inhibited the ATP-induced [Ca2+]i responses in the presence of nimodipine and ω-conotoxin. Cyanidin-3-glucoside also significantly inhibited KCl (50 mM)-induced [Ca2+]i increases. Cyanidin-3-glucoside significantly inhibited ATP-induced mitochondrial depolarization. The intracellular Ca2+ chelator BAPTA-AM or the mitochondrial Ca2+ uniporter inhibitor RU360 blocked the ATP-induced mitochondrial depolarization in the presence of cyanidin-3-glucoside. Cyanidin-3-glucoside blocked ATP-induced formation of ROS. BAPTA-AM further decreased the formation of ROS in the presence of cyanidin-3-glucoside. All these results suggest that cyanidin-3-glucoside inhibits ATP-induced calcium signaling in PC12 cells by inhibiting multiple pathways which are the influx of extracellular Ca2+ through the nimodipine and ω-conotoxin-sensitive and -insensitive pathways and the release of Ca2+ from intracellular stores. In addition, cyanidin-3-glucoside inhibits ATP-induced formation of ROS by inhibiting Ca2+-induced mitochondrial depolarization.