U-73122, an aminosteroid phospholipase C inhibitor, may also block Ca2+ influx through phospholipase C-independent mechanism in neutrophil activation

1-[6-[[17a-3-Methoxyestra-1,3,5(10)-trien17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122) has been proven to be a useful tool in investigation of phospholipase C (PLC)-coupled signal transduction during cell activation. In the present studies, the inhibition by U-73122 of cytosolic free Ca²⁺ concentration ([Ca 2+]i) of neutrophils was investigated. U-73122 suppressed the [Ca²⁺]i elevation of neutrophils suspended in Ca²⁺-containing medium challenged by N-formyl-Met-Leu-Phe (fMLP), cyclopiazonic acid (CPA) and ionomycin. The concentrations of U-73122 required for inhibition of CPA- and ionomycin-induced changes with IC₅₀ values 4.06 ± 0.27 µM and 4.04 ± 0.44 µM, respectively, is almost 10-times that required for inhibition of the fMLP-induced response (IC₅₀ value 0.62 ± 0.04 µM) U-73122 also reduced the intracellular Ca²⁺ mobilization of neutrophils suspended in Ca ²⁺-free medium stimulated by fMLP and CPA, but not by ionomycin, with IC₅₀ values 0.52 ± 0.02 µM and 6.82 ± 0.74 µM, respectively. 1-[6-[[17f3-Methoxyestra-1,3,5(10)-trien-l7-yl]amino]hexyl]2,5-pyrrolidinedione (U-73343), a close analog of U-73122 that does not inhibit PLC activity, suppressed the [Ca²⁺]_i elevation of neutrophils challenged by fMLP in Ca²⁺-containing medium, but not in Ca²⁺-free medium, with IC₅₀ value 22.30 ± 1.61 µM. In Mn²⁺-quench studies, U-73122 suppressed the Mn²⁺ influx in CPA-activated neutrophils (IC₅₀ value was 7.16 ± 0.28 µM) as well as in resting neutrophils (IC₅₀ value was 6.72 ± 0.30 M). U-73343 also suppressed the Mn²⁺ influx in resting neutrophils in a concentration-dependent manner. These results suggest that the inhibitory effect of U-73122 on [Ca²⁺]i of activated neutrophils is attributed partly to the suppression of Ca²⁺ release from the intracellular Ca²⁺ stores through PLC inhibition, and partly to the blockade, especially at higher concentrations, of Ca²⁺ entry from the extracellular space through PLC-independent processes.     

Endothelin-1 induced contraction of rat aorta: contributions made by Ca2+ influx and activation of contractile apparatus associated with no change in cytoplasmic Ca2+ level

Summary The modes by which Endothelin-1 (ET) induces Ca²⁺-influx and the relative functional importance of the different sources of Ca²⁺ for ET-induced contraction were studied using fura 2-loaded and unloaded rat aortic strips. ET caused an increase in the cytosolic free Ca²⁺ level ([Ca²⁺]_i) followed by a tonic contraction in Ca²⁺-containing solution, and produced a transient elevation of [Ca²⁺]_i followed by a small sustained contraction in Ca²⁺-free medium. ET also stimulated ⁴⁵Ca influx into La²⁺-inaccessible fraction significantly. With the same change of [Ca²⁺]_i, ET caused a larger tension than that induced by high K. ET-induced contraction and [Ca²⁺]_i elevation were not significantly inhibited by 0.1–0.3 M nicardipine which nearly abolished the contraction and [Ca⁺]_i elevation produced by high K. During treatment of the strips with high K, addition of ET induced further increases in [Ca²⁺]_i and muscle tension, and vice versa. In Ca²⁺-free medium, ET-induced contraction was influenced neither by ryanodine-treatment nor by high K-treatment, although the former attenuated and the latter potentiated the [Ca²⁺]_i transient induced by ET. Further, the ET-induced sustained contraction under Ca²⁺-free conditions began to develop after the [Ca²⁺]_i level returned to the baseline. Thus, it seems that the Ca²⁺ released from the ryanodine-sensitive and -insensitive Ca²⁺ stores by ET may provide only a minor or indirect contribution, if any, to the tension development. ET might cause a contraction mainly by stimulating Ca²⁺-influx through Ca²⁺ channel(s) other than voltage-dependent Ca²⁺ channels in character, and by increasing the sensitivity of the contractile filaments to Ca²⁺ or activating them Ca²⁺-independently.Visiting from Zun Yi Medical College, China Send offprint requests to I. Takayanagi at the above address     

Forskolin Changes the Relationship between Cytosolic Ca2+ and Contraction in Guinea Pig Ileum

This study was designed to clarify the mechanism of the inhibitory effect of forskolin on contraction, cytosolic Ca²⁺ level ([Ca²⁺]_i), and Ca²⁺ sensitivity in guinea pig ileum. Forskolin (0.1 nM~10 µM) inhibited high K⁺ (25 mM and 40 mM)- or histamine (3 µM)-evoked contractions in a concentration-dependent manner. Histamine-evoked contractions were more sensitive to forskolin than high K⁺-evoked contractions. Spontaneous changes in [Ca²⁺]_i and contractions were inhibited by forskolin (1 µM) without changing the resting [Ca²⁺]_i. Forskoln (10 µM) inhibited muscle tension more strongly than [Ca²⁺]_i stimulated by high K⁺, and thus shifted the [Ca²⁺]_i-tension relationship to the lower-right. In histamine-stimulated contractions, forskolin (1 µM) inhibited both [Ca²⁺]_i and muscle tension without changing the [Ca²⁺]_i-tension relationship. In α-toxin-permeabilized tissues, forskolin (10 µM) inhibited the 0.3 µM Ca²⁺-evoked contractions in the presence of 0.1 mM GTP, but showed no effect on the Ca²⁺-tension relationship. We conclude that forskolin inhibits smooth muscle contractions by the following two mechanisms: a decrease in Ca²⁺ sensitivity of contractile elements in high K⁺-stimulated muscle and a decrease in [Ca²⁺]_i in histamine-stimulated muscle.     

Brief low [Mg2+]o-induced Ca2+ spikes inhibit subsequent prolonged exposure-induced excitotoxicity in cultured rat hippocampal neurons

Reducing [Mg²⁺]_o to 0.1 mM can evoke repetitive [Ca²⁺]_i spikes and seizure activity, which induces neuronal cell death in a process called excitotoxicity. We examined the issue of whether cultured rat hippocampal neurons preconditioned by a brief exposure to 0.1 mM [Mg²⁺]_o are rendered resistant to excitotoxicity induced by a subsequent prolonged exposure and whether Ca²⁺ spikes are involved in this process. Preconditioning by an exposure to 0.1 mM [Mg²⁺]_o for 5 min inhibited significantly subsequent 24 h exposure-induced cell death 24 h later (tolerance). Such tolerance was prevented by both the NMDA receptor antagonist D-AP5 and the L-type Ca²⁺ channel antagonist nimodipine, which blocked 0.1 mM [Mg²⁺]_o-induced [Ca²⁺]_i spikes. The AMPA receptor antagonist NBQX significantly inhibited both the tolerance and the [Ca²⁺]_i spikes. The intracellular Ca²⁺ chelator BAPTA-AM significantly prevented the tolerance. The nonspecific PKC inhibitor staurosporin inhibited the tolerance without affecting the [Ca²⁺]_i spikes. While Gö6976, a specific inhibitor of PKCα had no effect on the tolerance, both the PKCε translocation inhibitor and the PKCζ pseudosubstrate inhibitor significantly inhibited the tolerance without affecting the [Ca²⁺]_i spikes. Furthermore, JAK-2 inhibitor AG490, MAPK kinase inhibitor PD98059, and CaMKII inhibitor KN-62 inhibited the tolerance, but PI-3 kinase inhibitor LY294,002 did not. The protein synthesis inhibitor cycloheximide significantly inhibited the tolerance. Collectively, these results suggest that low [Mg²⁺]_o preconditioning induced excitotoxic tolerance was directly or indirectly mediated through the [Ca²⁺]_i spike-induced activation of PKCε and PKCξ, JAK-2, MAPK kinase, CaMKII and the de novo synthesis of proteins.     

Stimulation of cellular free Ca2+ elevation and inhibition of store-operated Ca2+ entry by kazinol B in neutrophils

Kazinol B, a natural isoprenylated flavan, stimulated the [Ca²⁺]_i elevation in the presence or absence of Ca²⁺ in the medium. Treatment with chymotrypsin or phorbol 12-myristate 13-acetate to shedding of l-selectin had no effect on subsequent kazinol B-induced Ca²⁺ response. Upon initial cyclopiazonic acid (CPA) treatment in the absence of external Ca²⁺, the subsequent [Ca²⁺]_i rise followed by challenge with kazinol B was greatly diminished. The ryanodine receptor blockers, 8-bromo-cyclic ADP-ribose and ruthenium red did not affect kazinol B-evoked Ca²⁺ release from internal stores. However, the inhibitors of sphingosine kinase, dimethylsphingosine, but not dihydrosphingosine, inhibited kazinol B-induced Ca²⁺ release. Kazinol B-induced [Ca²⁺]_i rise was not affected by two nitric oxidase inhibitors, N-(3-aminomethyl)benzylacetamidine (1400W) and 7-nitroindazole, cytochalasin B and Na⁺-deprivation. This response was slightly attenuated by 2-aminoethyldiphenyl borate (2-APB), a d-myo-inositol 1,4,5-trisphosphate (IP₃) receptor blocker, and by genistein, a general tyrosine kinase inhibitor. However, the Ca²⁺ response was greatly diminished by two actin filament reorganizers, calyculin A and jasplakinolide, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), an inhibitor of phosphoinositide 3-kinase, N-(3-aminomethyl)benzylacetamidine (SB 203580), the p38 mitogen-activated protein kinase inhibitor, 1-[6-[17-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122), the inhibitor of phospholipase C-coupled processes, and by 0.3 mM La³⁺ or Ni²⁺. Kazinol B did not evoke any appreciable Ba²⁺ and Sr²⁺ entry into cells. The Ca²⁺ entry blockers, 1-[-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole (SKF-96365), but not cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12,330A), inhibited a kazinol B-induced [Ca²⁺]_i rise. Kazinol B had no effect on the pharmacologically isolated plasma membrane Ca²⁺-ATPase activity. In a Ca²⁺-free medium, kazinol B inhibited the subsequent Ca²⁺ addition, resulting in robust entry in CPA- and formyl peptide-activated cells. Kazinol B produced a concentration-dependent reduction in the mitochondrial membrane potential. These results indicate that kazinol B stimulates Ca²⁺ release from internal Ca²⁺ store, probably through the sphingosine 1-phosphate and IP₃ signaling, and activates external Ca²⁺ influx mainly through a non-store-operated Ca²⁺ entry (non-SOCE) pathway. Inhibition of SOCE by kazinol B is probably attributable to a break in the Ca²⁺ driven force of mitochondria.     

Hydrogen peroxide mobilizes Ca2+ through two distinct mechanisms in rat hepatocytes

Aim:

Hydrogen peroxide (H₂O₂) is produced during liver transplantation. Ischemia/reperfusion induces oxidation and causes intracellular Ca²⁺ overload, which harms liver cells. Our goal was to determine the precise mechanisms of these processes.

Methods:

Hepatocytes were extracted from rats. Intracellular Ca²⁺ concentrations ([Ca²⁺]_i), inner mitochondrial membrane potentials and NAD(P)H levels were measured using fluorescence imaging. Phospholipase C (PLC) activity was detected using exogenous PIP₂. ATP concentrations were measured using the luciferin-luciferase method. Patch-clamp recordings were performed to evaluate membrane currents.

Results:

H₂O₂ increased intracellular Ca²⁺ concentrations ([Ca²⁺]_i) across two kinetic phases. A low concentration (400 μmol/L) of H₂O₂ induced a sustained elevation of [Ca²⁺]_i that was reversed by removing extracellular Ca²⁺. H₂O₂ increased membrane currents consistent with intracellular ATP concentrations. The non-selective ATP-sensitive cation channel blocker amiloride inhibited H₂O₂-induced membrane current increases and [Ca²⁺]_i elevation. A high concentration (1 mmol/L) of H₂O₂ induced an additional transient elevation of [Ca²⁺]_i, which was abolished by the specific PLC blocker {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 but was not eliminated by removal of extracellular Ca²⁺. PLC activity was increased by 1 mmol/L H₂O₂ but not by 400 μmol/L H₂O₂.

Conclusion:

H₂O₂ mobilizes Ca²⁺ through two distinct mechanisms. In one, 400 μmol/L H₂O₂-induced sustained [Ca²⁺]_i elevation is mediated via a Ca²⁺ influx mechanism, under which H₂O₂ impairs mitochondrial function via oxidative stress, reduces intracellular ATP production, and in turn opens ATP-sensitive, non-specific cation channels, leading to Ca²⁺ influx. In contrast, 1 mmol/L H₂O₂-induced transient elevation of [Ca²⁺]_i is mediated via activation of the PLC signaling pathway and subsequently, by mobilization of Ca²⁺ from intracellular Ca²⁺ stores.     

Oxidized Low-density Lipoprotein- and Lysophosphatidylcholine-induced Ca2+ Mobilization in Human Endothelial Cells

The effects of oxidized low-density lipoprotein (OxLDL) and its major lipid constituent lysophosphatidylcholine (LPC) on Ca²⁺ entry were investigated in cultured human umbilical endothelial cells (HUVECs) using fura-2 fluorescence and patch-clamp methods. OxLDL or LPC increased intracellular Ca²⁺ concentration ([Ca²⁺]_i), and the increase of [Ca²⁺]_i by OxLDL or by LPC was inhibited by La³⁺ or heparin. LPC failed to increase [Ca²⁺]_i in the presence of an antioxidant tempol. In addition, store-operated Ca²⁺ entry (SOC), which was evoked by intracellular Ca²⁺ store depletion in Ca²⁺-free solution using the sarcoplasmic reticulum Ca²⁺ pump blocker, 2, 5-di-t-butyl-1, 4-benzohydroquinone (BHQ), was further enhanced by OxLDL or by LPC. Increased SOC by OxLDL or by LPC was inhibited by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122. In voltage-clamped cells, OxLDL or LPC increased [Ca²⁺]_i and simultaneously activated non-selective cation (NSC) currents. LPC-induced NSC currents were inhibited by 2-APB, La³⁺ or {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, and NSC currents were not activated by LPC in the presence of tempol. Furthermore, in voltage-clamped HUVECs, OxLDL enhanced SOC and evoked outward currents simultaneously. Clamping intracellular Ca²⁺ to 1 µM activated large-conductance Ca²⁺-activated K⁺ (BK_Ca) current spontaneously, and this activated BK_Ca current was further enhanced by OxLDL or by LPC. From these results, we concluded that OxLDL or its main component LPC activates Ca²⁺-permeable Ca²⁺-activated NSC current and BK_Ca current simultaneously, thereby increasing SOC.     

Effect of bisphenol A on Ca2+ fluxes and viability in Madin-Darby canine renal tubular cells

The effect of the environmental contaminant, bisphenol A, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in Madin-Darby canine kidney (MDCK) cells is unclear. This study explored whether bisphenol A changed basal [Ca²⁺]_i levels in suspended MDCK cells by using fura-2 as a Ca²⁺-sensitive fluorescent dye. Bisphenol A, at concentrations between 50 and 300 µM, increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced, partly, by removing extracellular Ca²⁺. Bisphenol A induced Mn²⁺ influx, leading to quenching of fura-2 fluorescence, suggesting Ca²⁺ influx. This Ca²⁺ influx was inhibited by phospholipase A2 inhibitor aristolochic acid, store-operated Ca²⁺ channel blockers nifedipine and SK&F96365, and protein kinase C inhibitor GF109203X. In Ca²⁺-free medium, pretreatment with the mitochondrial uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP), and the endoplasmic reticulum Ca²⁺ pump inhibitors, thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ), inhibited bisphenol A–induced Ca²⁺ release. Conversely, pretreatment with bisphenol A abolished thapsigargin (or BHQ)- and CCCP-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished bisphenol-induced [Ca²⁺]_i rise. Bisphenol A caused a concentration-dependent decrease in cell viability via apoptosis in a Ca²⁺-independent manner. Collectively, in MDCK cells, bisphenol A induced [Ca²⁺]_i rises by causing phospholipase C–dependent Ca²⁺ release from the endoplasmic reticulum and mitochondria and Ca²⁺ influx via phospholipase A2–, protein kinase C–sensitive, store-operated Ca²⁺ channels.     

Effect of calmidazolium on [Ca2+]i and viability in human hepatoma cells

The effect of calmidazolium on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability has not been explored in human hepatoma cells. This study examined whether calmidazolium altered [Ca²⁺]_i and caused cell death in HA59T cells. [Ca²⁺]_i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Calmidazolium at concentrations ≥1 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 1.5 μM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Calmidazolium induced Mn²⁺ quench of fura-2 fluorescence implicating Ca²⁺ influx. The Ca²⁺ influx was insensitive to L-type Ca²⁺ entry blockers, but was inhibited partly by enhancing or inhibiting protein kinase C activity. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), calmidazolium-induced [Ca²⁺]_i rises were largely inhibited; and conversely, calmidazolium pretreatment totally suppressed thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change calmidazolium-induced [Ca²⁺]_i rises. At concentrations between 1 and 15 μM, calmidazolium induced apoptosis-mediated cell death. Collectively, in HA59T hepatoma cells, calmidazolium induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx via protein kinase C-regulated Ca²⁺ entry pathway. Calmidazolium caused cytotoxicity via apoptosis.     

Effects of timolol on Ca2+ handling and viability in human prostate cancer cells

Timolol is a medication used widely to treat glaucoma. Regarding Ca²⁺ signaling, timolol was shown to modulate Ca²⁺-related physiology in various cell types, however, the effect of timolol on Ca²⁺ homeostasis and cell viability has not been explored in human prostate cancer cells. The aim of this study was to explore the effect of timolol on intracellular Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells. Timolol at concentrations of 100–1000?μM induced [Ca²⁺]_i rises. The Ca²⁺ signal in Ca²⁺-containing medium was reduced by removal of extracellular Ca²⁺ by approximately 75%. Timolol (1000?μM) induced Mn²⁺ influx suggesting of Ca²⁺ entry. Timolol-induced Ca²⁺ entry was partially inhibited by three inhibitors of store-operated Ca²⁺ channels: nifedipine, econoazole and SKF96365, and by a protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate [PMA]) or an inhibitor (GF109203X). In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin abolished timolol-evoked [Ca²⁺]_i rises. Conversely, treatment with timolol abolished thapsigargin-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 abolished timolol-induced [Ca²⁺]_i rises. Timolol at concentrations between 200 and 600?μM killed cells in a concentration-dependent fashion. Chelation of cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not reverse cytotoxicity of timolol. Together, in PC3 cells, timolol induced [Ca²⁺]_i rises by evoking Ca²⁺release from the endoplasmic reticulum in a PLC-dependent manner, and Ca²⁺ influx via PKC-regulated store-operated Ca²⁺ entry. Timolol also caused cell death that was not linked to preceding [Ca²⁺]_i rises.     

Effect of celecoxib on Ca2+ handling and viability in human prostate cancer cells (PC3)

Celecoxib has been shown to have an antitumor effect in previous studies, but the mechanisms are unclear. Ca²⁺ is a key second messenger in most cells. The effect of celecoxib on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in human suspended PC3 prostate cancer cells was explored by using fura-2 as a fluorescent dye. Celecoxib at concentrations between 5 and 30 μM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Celecoxib-induced Ca²⁺ influx was not blocked by L-type Ca²⁺ entry inhibitors or protein kinase C/A modulators [phorbol 12-myristate 13-acetate (PMA), GF109203X, H-89], but was inhibited by the phospholipase A₂ inhibitor, aristolochic acid. In Ca²⁺-free medium, 30 μM of celecoxib failed to induce a [Ca²⁺]_i rise after pretreatment with thapsigargin (an endoplasmic reticulum [ER] Ca²⁺ pump inhibitor). Conversely, pretreatment with celecoxib inhibited thapsigargin-induced Ca²⁺ release. Inhibition of phospholipase C with U73122 did not change celecoxib-induced [Ca²⁺]_i rises. Celecoxib induced slight cell death in a concentration-dependent manner, which was enhanced by chelating cytosolic Ca²⁺ with BAPTA. Collectively, in PC3 cells, celecoxib induced [Ca²⁺]_i rises by causing phospholipase C–independent Ca²⁺ release from the ER and Ca²⁺ influx via non-L-type, phospholipase A₂-regulated Ca²⁺ channels. These data may contribute to the understanding of the effect of celecoxib on prostate cancer cells.     

Effect of thymol on Ca homeostasis and viability in human glioblastoma cells

The effect of the natural essential oil thymol on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in human glioblastoma cells was examined. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Thymol at concentrations of 400–1000 μM induced a [Ca²⁺]_i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca²⁺. Thymol-induced Ca²⁺ signal was not altered by nifedipine, econazole, SK&F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca²⁺ was removed, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished thymol-induced [Ca²⁺]_i rise. Incubation with thymol also abolished thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished thymol-induced [Ca²⁺]_i rise. At concentrations of 200–800 μM, thymol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that thymol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. Collectively, in human glioblastoma cells, thymol induced a [Ca²⁺]_i rise by inducing phospholipase C- and protein kinase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via non store-operated Ca²⁺ channels. Thymol induced cell death that may involve apoptosis.     

Mechanisms of AM404-induced [Ca]i rise and death in human osteosarcoma cells

The effect of N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca²⁺ levels ([Ca²⁺]_i) and viability was studied in human MG63 osteosarcoma cells using the fluorescent dyes fura-2 and WST-1, respectively. AM404 at concentrations ≥5 μM increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 60 μM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. AM404 induced Mn²⁺ quench of fura-2 fluorescence implicating Ca²⁺ influx. The Ca²⁺ influx was sensitive to La³⁺, Ni²⁺, nifedipine and verapamil. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), AM404-induced [Ca²⁺]_i rise was abolished; and conversely, AM404 pretreatment totally inhibited thapsigargin-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not change AM404-induced [Ca²⁺]_i rise. At concentrations between 10 and 200 μM, AM404 killed cells in a concentration-dependent manner presumably by inducing apoptotic cell death. The cytotoxic effect of 50 μM AM404 was partly reversed by prechelating cytosolic Ca²⁺ with BAPTA/AM. Collectively, in MG63 cells, AM404 induced [Ca²⁺]_i rise by causing Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx via L-type Ca²⁺ channels. AM404 caused cytotoxicity which was possibly mediated by apoptosis.     

Ca2+ entry following P2X receptor activation induces IP3 receptor-mediated Ca2+ release in myocytes from small renal arteries

BACKGROUND AND PURPOSE

P2X receptors mediate sympathetic control and autoregulation of the renal circulation triggering contraction of renal vascular smooth muscle cells (RVSMCs) via an elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i). Although it is well-appreciated that the myocyte Ca²⁺ signalling system is composed of microdomains, little is known about the structure of the [Ca²⁺]_i responses induced by P2X receptor stimulation in vascular myocytes.

EXPERIMENTAL APPROACHES

Using confocal microscopy, perforated-patch electrical recordings, immuno-/organelle-specific staining, flash photolysis and RT-PCR analysis we explored, at the subcellular level, the Ca²⁺ signalling system engaged in RVSMCs on stimulation of P2X receptors with the selective agonist αβ-methylene ATP (αβ-meATP).

KEY RESULTS

RT-PCR analysis of single RVSMCs showed the presence of genes encoding inositol 1,4,5-trisphosphate receptor type 1(IP₃R1) and ryanodine receptor type 2 (RyR2). The amplitude of the [Ca²⁺]_i transients depended on αβ-meATP concentration. Depolarization induced by 10 µmol·L⁻¹αβ-meATP triggered an abrupt Ca²⁺ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum enriched with IP₃Rs but poor in RyRs. Depletion of calcium stores, block of voltage-gated Ca²⁺ channels (VGCCs) or IP₃Rs suppressed the sub-plasmalemmal [Ca²⁺]_i upstroke significantly more than block of RyRs. The effect of calcium store depletion or IP₃R inhibition on the sub-plasmalemmal [Ca²⁺]_i upstroke was attenuated following block of VGCCs.

CONCLUSIONS AND IMPLICATIONS

Depolarization of RVSMCs following P2X receptor activation induces IP₃R-mediated Ca²⁺ release from sub-plasmalemmal (‘junctional’) sarcoplasmic reticulum, which is activated mainly by Ca²⁺ influx through VGCCs. This mechanism provides convergence of signalling pathways engaged in electromechanical and pharmacomechanical coupling in renal vascular myocytes.     

Ketamine attenuates the Na+-dependent Ca2+ overload in rabbit ventricular myocytes in vitro by inhibiting late Na+ and L-type Ca2+ currents

Aim:

Intracellular Ca²⁺ ([Ca²⁺]_i) overload occurs in myocardial ischemia. An increase in the late sodium current (I_NaL) causes intracellular Na⁺ overload and subsequently [Ca²⁺]_i overload via the reverse-mode sodium-calcium exchanger (NCX). Thus, inhibition of INaL is a potential therapeutic target for cardiac diseases associated with [Ca²⁺]_i overload. The aim of this study was to investigate the effects of ketamine on Na⁺-dependent Ca²⁺ overload in ventricular myocytes in vitro.

Methods:

Ventricular myocytes were enzymatically isolated from hearts of rabbits. I_NaL, NCX current (I_NCX) and L-type Ca²⁺ current (I_CaL) were recorded using whole-cell patch-clamp technique. Myocyte shortening and [Ca²⁺]_i transients were measured simultaneously using a video-based edge detection and dual excitation fluorescence photomultiplier system.

Results:

Ketamine (20, 40, 80 μmol/L) inhibited I_NaL in a concentration-dependent manner. In the presence of sea anemone toxin II (ATX, 30 nmol/L), I_NaL was augmented by more than 3-fold, while ketamine concentration-dependently suppressed the ATX-augmented I_NaL. Ketamine (40 μmol/L) also significantly suppressed hypoxia or H₂O₂-induced enhancement of I_NaL. Furthermore, ketamine concentration-dependently attenuated ATX-induced enhancement of reverse-mode I_NCX. In addition, ketamine (40 μmol/L) inhibited I_CaL by 33.4%. In the presence of ATX (3 nmol/L), the rate and amplitude of cell shortening and relaxation, the diastolic [Ca²⁺]_i, and the rate and amplitude of [Ca²⁺]_i rise and decay were significantly increased, which were reverted to control levels by tetrodotoxin (TTX, 2 μmol/L) or by ketamine (40 μmol/L).

Conclusion:

Ketamine protects isolated rabbit ventricular myocytes against [Ca²⁺]_i overload by inhibiting I_NaL and I_CaL.     

The garlic ingredient diallyl sulfide induces Ca mobilization in Madin-Darby canine kidney cells

Diallyl sulfide (DAS), one of the major organosulfur compounds (OSCs) of garlic, is recognized as a group of potential chemoproventive compounds. In this study, we examines the early signaling effects of DAS on renal cells loaded with Ca²⁺-sensitive dye fura-2. It was found that DAS caused an immediate and sustained rise of [Ca²⁺]_i in a concentration-dependent manner (EC₅₀ = 2.32 mM). DAS also induced a [Ca²⁺]_i elevation when extracellular Ca²⁺ was removed, but the magnitude was reduced by 45%. Depletion of intracellular Ca²⁺ stores with CCCP, a mitochondrial uncoupler, did not affect DAS’s effect. In Ca²⁺-free medium, the DAS-induced [Ca²⁺]_i rise was abolished by depleting stored Ca²⁺ with thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor). DAS-caused [Ca²⁺]_i rise in Ca²⁺-containing medium was not affected by modulation of protein kinase C activity. The DAS-induced Ca²⁺ influx was blocked by nicardipine. U73122, an inhibitor of phospholipase C, abolished ATP (but not DAS)-induced [Ca²⁺]_i rise. Additionally, pretreatment with DAS for 24 h decreased cell viability in a concentration-dependent manner. Furthermore, DAS-induced cell death involved apoptotic events. These findings suggest that diallyl sulfide induced a significant rise in [Ca²⁺]_i in MDCK renal tubular cells by stimulating both extracellular Ca²⁺ influx and thapsigargin-sensitive intracellular Ca²⁺ release via as yet unidentified mechanisms.     

Effect of diallyl disulfide on Ca movement and viability in PC3 human prostate cancer cells

The effect of diallyl disulfide (DADS) on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells is unclear. This study explored whether DADS changed [Ca²⁺]_i in PC3 cells by using fura-2. DADS at 50-1000 μM increased [Ca²⁺]_i in a concentration-dependent manner. The signal was reduced by removing Ca²⁺. DADS-induced Ca²⁺ influx was not inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators; but was inhibited by aristolochic acid. In Ca²⁺-free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) nearly abolished DADS-induced [Ca²⁺]_i rise. Incubation with DADS inhibited thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not alter DADS-induced [Ca²⁺]_i rise. At 500-1000 μM, DADS killed cells in a concentration-dependent manner. The cytotoxic effect of DADS was partly reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Propidium iodide staining suggests that DADS (500 μM) induced apoptosis in a Ca²⁺-independent manner. Annexin V/PI staining further shows that 10 μM and 500 μM DADS both evoked apoptosis. DADS also increased reactive oxygen species (ROS) production. Collectively, in PC3 cells, DADS induced [Ca²⁺]_i rise probably by causing phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via phospholipase A₂-sensitive channels. DADS induced Ca²⁺-dependent cell death, ROS production, and Ca²⁺-independent apoptosis.     

Inhibition of store-operated Ca(2+) channels prevent ethanol-induced intracellular Ca(2+) increase and cell injury in a human hepatoma cell line

Elevated intracellular Ca²⁺ content is implicated in ethanol-induced hepatocyte apoptosis and necrosis. Extracellular Ca²⁺ influx has been suggested to play a role in this process. However, the exact Ca²⁺-permeable channel involved in the plasma membrane is still unclear. This study investigated the role of store-operated calcium entry (SOCE) in ethanol-induced cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) increase and hepatotoxicity. Ethanol (25-800 mM) dose-dependently increased [Ca²⁺]_i content and hepatocyte damage in HepG2 cells. 2-aminoethoxydiphenyl borate (2-APB), the proved efficient antagonist of SOCs, dose-dependently suppressed the ethanol (200 nM)-increased [Ca²⁺]_i content and protected against ethanol-induced viability loss and transaminase leakage. Exposure to 200 mM ethanol for 24 h significantly upregulated the mRNA and protein expression of calcium release-activated calcium channel protein 1 (CRACM1, Orai1) and stromal interaction molecule 1 (STIM1), the two main molecular constituents of SOCs, which was sustained for at least 72 h. In addition, small interfering RNA knockdown of STIM1 attenuated the ethanol-increased [Ca²⁺]_i content and hepatotoxicity. Taken together, these data indicate that the Ca²⁺ channel of SOCE may be involved in the pathogenesis of ethanol-induced intracellular Ca²⁺ elevation and consequent hepatocyte damage.     

Ca2+ is a Regulator of the WNK/OSR1/NKCC Pathway in a Human Salivary Gland Cell Line

Wnk kinase maintains cell volume, regulating various transporters such as sodium-chloride cotransporter, potassium-chloride cotransporter, and sodium-potassium-chloride cotransporter 1 (NKCC1) through the phosphorylation of oxidative stress responsive kinase 1 (OSR1) and STE20/SPS1-related proline/alanine-rich kinase (SPAK). However, the activating mechanism of Wnk kinase in specific tissues and specific conditions is broadly unclear. In the present study, we used a human salivary gland (HSG) cell line as a model and showed that Ca²⁺ may have a role in regulating Wnk kinase in the HSG cell line. Through this study, we found that the HSG cell line expressed molecules participating in the WNK-OSR1-NKCC pathway, such as Wnk1, Wnk4, OSR1, SPAK, and NKCC1. The HSG cell line showed an intracellular Ca²⁺ concentration ([Ca²⁺]_i) increase in response to hypotonic stimulation, and the response was synchronized with the phosphorylation of OSR1. Interestingly, when we inhibited the hypotonically induced [Ca²⁺]_i increase with nonspecific Ca²⁺ channel blockers such as 2-aminoethoxydiphenyl borate, gadolinium, and lanthanum, the phosphorylated OSR1 level was also diminished. Moreover, a cyclopiazonic acid-induced passive [Ca²⁺]_i elevation was evoked by the phosphorylation of OSR1, and the amount of phosphorylated OSR1 decreased when the cells were treated with BAPTA, a Ca²⁺ chelator. Finally, through that process, NKCC1 activity also decreased to maintain the cell volume in the HSG cell line. These results indicate that Ca²⁺ may regulate the WNK-OSR1 pathway and NKCC1 activity in the HSG cell line. This is the first demonstration that indicates upstream Ca²⁺ regulation of the WNK-OSR1 pathway in intact cells.     

Calcium mobilization induced by endothelins and sarafotoxin in cultured canine tracheal smooth muscle cells

Endothelins (ETs)- and sarafotoxin (S6b)-induced rises in intracellular Ca²⁺ concentration ([Ca²⁺]_i) were monitored in cultured canine tracheal smooth muscle cells by using a fluorescent Ca²⁺ indicator fura-2. ET-1, ET-2, ET-3 and S6b elicited an initial transient peak and followed by a sustained elevation of [Ca²⁺]_i, with half-maximal effect (EC₅₀) of 18, 20, 38 and 21 nM, respectively. BQ-123, an ET_A receptor antagonist, had a high affinity to block the rise in [Ca2⁺]_i response to ET-1, ET-2, and S6b, as well as a low affinity for ET-3. Removal of external Ca²⁺ by addition of EGTA during the sustained phase, caused a rapid decline in [Ca²⁺]_i to the resting level. In the absence of external Ca²⁺, only an initial transient peak of [Ca²⁺]_i was seen, the sustained elevation of [Ca²⁺]. could then be evoked by addition of 1.8 mM Ca²⁺. Ca²⁺ influx was required for the changes of [Ca²⁺]_i, since the Ca²⁺-channel blockers, diltiazem, verapamil, and Ni²⁺, decreased both the initial and sustained elevation of [Ca²⁺]_i response to these peptides. ETs exhibited homologous desensitization of the Ca²⁺ response, but partial heterologous desensitization of the Ca²⁺ response mediated by carbachol to different extents. In contrast, ETs did not desensitize the Ca²⁺ response induced by ATP or vice versa. These data demonstrate that the initial detectable increase in [Ca2⁺]_i stimulated by these peptides is due to the activation of ET_A receptors and subsequently the release of Ca²⁺ from internal stores, whereas the contribution of external Ca²⁺ follows and partially involves a diltiazem- and verapamil-sensitive process. There is a cross-regulation among ETs and other receptor-coupling signal transduction pathways through PI hydrolysis in canine tracheal smooth muscle cells. Correspondence to: C. Mao Yang at the above address