Cryptogein-induced initial events in tobacco BY-2 cells: pharmacological characterization of molecular relationship among cytosolic Ca(2+) transients, anion efflux and production of reactive oxygen species

Ion fluxes and the production of reactive oxygen species (ROS) are early events that follow elicitor treatment or microbial infection. However, molecular mechanisms for these responses as well as their relationship have been controversial and still largely unknown. We here simultaneously monitored the temporal sequence of initial events at the plasma membrane in suspension-cultured tobacco cells (cell line BY-2) in response to a purified proteinaceous elicitor, cryptogein, which induced hypersensitive cell death. The elicitor induced transient rise in cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) showing two distinct peaks, followed by biphasic (rapid/transient and slow/prolonged) Cl(-) efflux and H(+) influx. Pharmacological analyses suggested that the two phases of the [Ca(2+)](cyt) response correspond to Ca(2+) influx through the plasma membrane and an inositol 1,4,5-trisphophate-mediated release of Ca(2+) from intracellular Ca(2+) stores, respectively, and the [Ca(2+)](cyt) transients and the Cl(-) efflux were mutually dependent events regulated by protein phosphorylation. The elicitor also induced production of ROS including (*)O(2)(-) and H(2)O(2), which initiated after the [Ca(2+)](cyt) rise and required Ca(2+) influx, Cl(-) efflux and protein phosphorylation. An inhibitor of NADPH oxidase, diphenylene iodonium, completely inhibited the elicitor-induced production of (*)O(2)(-) and H(2)O(2), but did not affect the [Ca(2+)](cyt) transients. These results suggest that cryptogein-induced plasma membrane Ca(2+) influx is independent of ROS, and NADPH oxidase dependent ROS production is regulated by these series of ion fluxes.     

Effect of hydrogen peroxide on reoxygenation-induced Ca2+ accumulation in rat cardiomyocytes

Reactive oxygen species (ROS) contribute to cell damage during reperfusion of the heart. ROS may exert their effects partly by interfering with Ca(2+) homeostasis of the myocardium. The purpose of this study was to investigate the effects of hydrogen peroxide (H(2)O(2)) on Ca(2+) accumulation during reoxygenation of isolated adult rat cardiomyocytes exposed to 1 h of hypoxia and to relate the effects to possible changes in release of lactate dehydrogenase (LDH), free intracellular Ca(2+) ([Ca(2+)](i)) and Mg(2+)([Mg(2+)](i)), and mitochondrial membrane potential (Deltapsim). Cell Ca(2+) was determined by (45)Ca(2+) uptake. Free [Mg(2+)](i) and [Ca(2+)](i) and Deltapsim were measured by flow cytometry. Reoxygenation-induced Ca(2+) accumulation was attenuated by 23 and 34% by 10 and 25 microM H(2)O(2), respectively, added at reoxygenation. H(2)O(2) at 100 and 250 microM increased cell Ca(2+) by 50 and 83%, respectively, whereas 500 microM H(2)O(2) decreased cell Ca(2+) by 20%. H(2)O(2) at (25 microM) reduced LDH release and [Mg(2+)](i) and increased Deltapsim, indicating cell protection, whereas 250 microM H(2)O(2) increased LDH release and [Mg(2+)](i) and decreased Deltapsim, indicating cell damage. Clonazepam (100 microM) attenuated the increase in Ca(2+) accumulation, the elevation of [Ca(2+)](i), and the decrease in Deltapsim induced by 100 and 250 microM H(2)O(2) during reoxygenation. We report for the first time that 25 microM H(2)O(2) attenuates Ca(2+) accumulation, LDH release, and dissipation of Deltapsim during reoxygenation of hypoxic cardiomyocytes, indicating cell protection.     

Reactive oxygen species stimulated human hepatoma cell proliferation via cross-talk between PI3-K/PKB and JNK signaling pathways

Reactive oxygen species (ROS) are important for intracellular signaling mechanisms regulating many cellular processes. Manganese superoxide dismutase (MnSOD) may regulate cell growth by changing the level of intracellular ROS. In our study, we investigated the effect of ROS on 7721 human hepatoma cell proliferation. Treatment with H2O2 (1-10 microM) or transfection with antisense MnSOD cDNA constructs significantly increased the cell proliferation. Recently, the mitogen-activated protein kinases (MAPK) and the protein kinase B (PKB) were proposed to be involved in cell growth. Accordingly, we assessed the ability of ROS to activate MAPK and PKB. PKB and extracellular signal-regulated kinase (ERK) were both rapidly and transiently activated by 10 microM H2O2, but the activities of p38 MAPK and JNK were not changed. ROS-induced PKB activation was abrogated by the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002, suggesting that PI3-K is an upstream mediator of PKB activation in 7721 cells. Transfection with sense PKB cDNA promoted c-fos and c-jun expression in 7721 cells, suggesting that ROS may regulate c-fos and c-jun expression via the PKB pathway. Furthermore we found that exogenous H2O2 could stimulate the proliferation of PKB-AS7721 cells transfected with antisense PKB cDNA, which was partly dependent on JNK activation, suggesting that H2O2 stimulated hepatoma cell proliferation via cross-talk between the PI3-K/PKB and the JNK signaling pathways. However, insulin could stimulate 7721 cell proliferation, which is independent of cross-talk between PI3-K/PKB and JNK pathways. In addition, H2O2 did not induce the cross-talk between the PI3-K/PKB and the JNK pathways in normal liver cells. Taken together, we found that ROS regulate hepatoma cell growth via specific signaling pathways (cross-talk between PI3-K/PKB and JNK pathway) which may provide a novel clue to elucidate the mechanism of hepatoma carcinogenesis.     

Nitric oxide induces dose-dependent CA(2+) transients and causes temporal morphological hyperpolarization in human neutrophils

We exposed adherent neutrophils to the nitric oxide (NO)-radical donors S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), and sodium nitroprusside (SNP) to study the role of NO in morphology and Ca(2+) signaling. Parallel to video imaging of cell morphology and migration in neutrophils, changes in intracellular free Ca(2+) ([Ca(2+)](i)) were assessed by ratio imaging of Fura-2. NO induced a rapid and persistent morphological hyperpolarization followed by migrational arrest that usually lasted throughout the 10-min experiments. Addition of 0.5-800 microM SNAP caused concentration-dependent elevation of [Ca(2+)](i) with an optimal effect at 50 microM. This was probably induced by NO itself, because no change in [Ca(2+)](i) was observed after treatment with NO donor byproducts, i.e. D-penicillamine, glutathione, or potassium cyanide. Increasing doses of SNAP (>/=200 microM) attenuated the Ca(2+) response to the soluble chemotactic stimulus formyl-methionyl-leucyl-phenylalanine (fMLP), and both NO- and fMLP-induced Ca(2+) transients were abolished at 800 microM SNAP or more. In kinetic studies of fluorescently labeled actin cytoskeleton, NO markedly reduced the F-actin content and profoundly increased cell area. Immunoblotting to investigate the formation of nitrotyrosine residues in cells exposed to NO donors did not imply nitrosylation, nor could we mimic the effects of NO with the cell permeant form of cGMP, i.e., 8-Br-cGMP. Hence these processes were probably not the principal NO targets. In summary, NO donors initially increased neutrophil morphological alterations, presumably due to an increase in [Ca(2+)](i), and thereafter inhibited such shape changes. Our observations demonstrate that the effects of NO donors are important for regulation of cellular signaling, i.e., Ca(2+) homeostasis, and also affect cell migration, e.g., through effects on F-actin turnover. Our results are discussed in relation to the complex mechanisms that govern basic cell shape changes, required for migration.     

Blockade of 5-HT(3) receptor with MDL7222 and Y25130 reduces hydrogen peroxide-induced neurotoxicity in cultured rat cortical cells

The present study was performed to examine the neuroprotective effects of 5-hydroxytryptamine (5-HT)(3) receptor antagonists against hydrogen peroxide (H(2)O(2))-induced neurotoxicity using cultured rat cortical neurons. Pretreatment of 5-HT(3) receptor antagonists, tropanyl-3,5-dichlorobenzoate (MDL72222, 0.1 and 1 microM) and N-(1-azabicyclo[2.2.2.]oct-3-yl)-6-chloro-4-ethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-8-carboxamide hydrochloride (Y25130, 0.5 and 5 microM), significantly inhibited the H(2)O(2) (100 microM)-induced neuronal cell death as assessed by a MTT assay and the number of apoptotic nuclei, evidenced by Hoechst 33342 staining. The protective effects of MDL72222 (1 microM) and Y25130 (5 microM) were completely blocked by the simultaneous treatment with 100 microM 1-phenylbiguanide, a 5-HT(3) receptor agonist, indicating that the protective effects of these compounds were due to 5-HT(3) receptor blockade. In addition, MDL72222 (1 microM) and Y25130 (5 microM) inhibited the H(2)O(2) (100 microM)-induced elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)) and glutamate release, generation of reactive oxygen species (ROS), and caspase-3 activity. These results suggest that the activation of the 5-HT(3) receptor may be partially involved in H(2)O(2)-induced neurotoxicity, by membrane depolarization for Ca(2+) influx. Therefore, the blockade of 5-HT(3) receptor with MDL72222 and Y25130 may ameliorate the H(2)O(2)-induced neurotoxicity by interfering with the increase of [Ca(2+)](c), and then by inhibiting glutamate release, generation of ROS and caspase-3 activity.     

Streptomycin and its analogues are potent inhibitors of the hypotonicity-induced Ca2+ entry and Cl- channel activity

Streptomycin is a common antibiotic used in culture media. It is also a known blocker of stretch-activated and mechanosensitive ion channels in neurons and cardiac myocytes. But very little information is available on its effect in the regulation of epithelial ion channels. Osmotic swelling is a kind of mechanical stretch. The opening of stretch-activated Ca(2+) channels contributes to hypotonicity-induced Ca(2+) influx which is necessary for the activation of volume-regulated Cl(-) channels in human cervical cancer cells. This study aimed to investigate the role of streptomycin in cell volume regulation. Treatment of cervical cancer SiHa cells with streptomycin and its analogues (gentamicin and netilmicin) did not affect the basal cytosolic Ca(2+) ([Ca(2+)](i)) level. But it attenuated the hypotonicity-stimulated increase of [Ca(2+)](i) in a dose-dependent manner with half-maximal inhibitory concentrations (IC(50)) of 25, 90 and 200 microM for streptomycin, gentamicin and netilmicin, respectively, when measured at room temperature. In contrast, under free extracellular Ca(2+) condition, hypotonic stress only induced a small, progressive increase of [Ca(2+)](i), while 500 microM streptomycin did not affect this Ca(2+) signaling. Streptomycin and its analogues (gentamicin and netilmicin) also inhibited the activation of volume-regulated Cl(-) channels in a dose-dependent manner with IC(50) of 30, 95 and 250 microM at room temperature, respectively. Chronic culture with 50 microM streptomycin downregulates the activity of volume-regulated Cl(-) channels and retards the process of regulatory volume decrease in SiHa cells and MDCK cells. We suggest that using cells chronically cultured with streptomycin to study epithelial ion channels risks studying cellular and molecular pathology rather than physiology.     

Effect of Bcl-x(L) overexpression on reactive oxygen species,intracellular calcium,and mitochondrial membrane potential following injury in astrocytes

Many studies have demonstrated the protective effects of Bcl-x(L) against both apoptotic and necrotic cell death, but the mode of action of Bcl-x(L) remains unclear. This work analyzed effects of Bcl-x(L) overexpression on cellular levels of reactive oxygen species (ROS), intracellular calcium ([Ca(2+)](i)), and mitochondrial membrane potential (DeltaPsi(m)) in cultured mouse primary astrocytes after exposure to glucose deprivation (GD) or hydrogen peroxide (H(2)O(2)). Upon exposure to GD or H(2)O(2), uninfected and Lac-Z-expressing astrocytes showed an immediate, rapid increase in ROS accumulation that was slowed and or reduced by Bcl-x(L). Changes in DeltaPsi(m) in response to the two insults differed. H(2)O(2) induced a decrease in DeltaPsi(m) that was initially greater in Bcl-x(L) cells, but then held stable. DeltaPsi(m) in control and Lac-Z-expressing cells initially declined more slowly, but after about 20 min showed rapid deterioration. Five hours of GD caused mitochondrial membrane hyperpolarization followed by a decrease in DeltaPsi(m,) which was not observed with Bcl-x(L) overexpression. Bcl-x(L) failed to inhibit the calcium dysregulation seen in control cells exposed to 400 microM H(2)O(2), but still improved cell survival. There was no increase in [Ca(2+)](i) with 5 h of GD. These data thus dissociate the effect of Bcl-x(L) on calcium homeostasis from effects on ROS, DeltaPsi(m,) and for H(2)O(2) exposure, cell survival.     

Effect of Y-24180 on Ca2+ movement and proliferation in renal tubular cells

In canine renal tubular cells, the effect of Y-24180, a presumed specific platelet activating factor (PAF) receptor antagonist, on intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by using fura-2 as a Ca(2+)-sensitive fluorescent probe. Y-24180 (0.1-10 microM) caused a rapid and sustained [Ca(2+)](i) rise in a concentration-dependent manner. The [Ca(2+)](i) rise was prevented by 30% by removal of extracellular Ca(2+), but was not changed by dihydropyridines, verapamil and diltiazem. Y-24180-induced Ca(2+) influx was confirmed by Mn(2+)-influx induced quench of fura-2 fluorescence. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of 5 microM Y-24180 on [Ca(2+)](i) was abolished; conversely, depletion of Ca(2+) stores with 5 microM Y-24180 abolished thapsigargin-induced [Ca(2+)](i) rise. U73122, an inhibitor of phoispholipase C, inhibited ATP-, but not Y-24180-induced [Ca(2+)](i) rise. Overnight treatment with Y-24180 did not alter cell proliferation rate. Collectively, these results suggest that Y-24180 acts as a potent, but not cytotoxic, Ca(2+) mobilizer in renal tubular cells, by stimulating both extracellular Ca(2+) influx and intracellular Ca(2+) release. Since alterations in Ca(2+) movement may interfere many cellular signaling processes unrelated to modulation of PAF receptors, caution must be applied in using this chemical as a selective PAF receptor antagonist.     

Effects of reactive oxygen species on proliferation of Chinese hamster lung fibroblast (V79) cells

Reactive oxygen species (ROS) have emerged as important signaling molecules in the regulation of various cellular processes. In our study, we investigated the effect of a wide range of ROS on Chinese hamster lung fibroblast (V79) cell proliferation. Treatment with H2O2 (100 microM), superoxide anion (generated by 1 mM xanthine and 1 mU/ml xanthine oxidase), menadione, and phenazine methosulfate increased the cell proliferation by approximately 50%. Moreover, a similar result was observed after partial inhibition of superoxide dismutase (SOD) and glutathione peroxidase. This upregulation of cell proliferation was suppressed by pretreatment with hydroxyl radical scavengers and iron chelating agents. In addition to ROS, treatment with exogenous catalase and SOD mimic (MnTMPyP) suppressed the normal cell proliferation. Short-term exposure of the cells to 100 microM H2O2 was sufficient to induce proliferation, which indicated that activation of the signaling pathway is important as an early event. Accordingly, we assessed the ability of H2O2 to activate mitogen-activated protein kinases (MAPK). Jun-N-terminal kinase (JNK) and p38 MAPK were both rapidly and transiently activated by 100 microM H2O2, with maximal activation 30 min after treatment. However, the activity of extracellular signal-regulated kinase (ERK) was not changed. Pretreatment with SB203580 and SB202190, specific inhibitors of p38 MAPK, reduced the cell proliferation induced by H2O2. The activation of both JNK and p38 MAPK was also suppressed by pretreatment with hydroxyl radical scavenger and iron chelating agents. Our results suggest that the trace metal-driven Fenton reaction is a central mechanism that underlies cell proliferation and MAPK activation.     

Hydrogen sulfide prevents hypoxia-induced apoptosis via inhibition of an H(2)O(2)-activated calcium signaling pathway in mouse hippocampal neurons

Hydrogen sulfide (H(2)S), an endogenous gaseous mediator, has been shown to exert protective effects against damage to different organs in the human body caused by various stimuli. However, the potential effects of H(2)S on hypoxia-induced neuronal apoptosis and its mechanisms remain unclear. Here, we exposed mouse hippocampal neurons to hypoxic conditions (2% O(2), 5% CO(2) and 93% N(2) at 37°C) to establish a hypoxic cell model. We found that 4-h hypoxia treatment significantly increased intracellular reactive oxygen species (ROS) levels, and pretreatment with NaHS (a source of H(2)S) for 30min suppressed hypoxia-induced intracellular ROS elevation. The hypoxia treatment significantly increased cytosolic calcium ([Ca(2+)](i)), and pretreatment with NaHS prevented the increase in [Ca(2+)](i). Additionally, polyethylene glycol (PEG)-catalase (a H(2)O(2) scavenger) but not PEG-SOD (an O(2)(-) scavenger) conferred an inhibitory effect similar to H(2)S on the hypoxia-induced increase in [Ca(2+)](i). Furthermore, we found that pretreatment with NaHS could significantly inhibit hypoxia-induced neuronal apoptosis, which was also inhibited by PEG-catalase or the inositol 1,4,5-triphosphate (IP(3)) receptor blocker xestospongin C. Taken together, these findings suggest that H(2)S inhibits hypoxia-induced apoptosis through inhibition of a ROS (mainly H(2)O(2))-activated Ca(2+) signaling pathway in mouse hippocampal neurons.     

Convergence of calcium signaling pathways of pathogenic elicitors and abscisic acid in Arabidopsis guard cells

A variety of stimuli, such as abscisic acid (ABA), reactive oxygen species (ROS), and elicitors of plant defense reactions, have been shown to induce stomatal closure. Our study addresses commonalities in the signaling pathways that these stimuli trigger. A recent report showed that both ABA and ROS stimulate an NADPH-dependent, hyperpolarization-activated Ca(2+) influx current in Arabidopsis guard cells termed "I(Ca)" (Z.M. Pei, Y. Murata, G. Benning, S. Thomine, B. Klüsener, G.J. Allen, E. Grill, J.I. Schroeder, Nature [2002] 406: 731-734). We found that yeast (Saccharomyces cerevisiae) elicitor and chitosan, both elicitors of plant defense responses, also activate this current and activation requires cytosolic NAD(P)H. These elicitors also induced elevations in the concentration of free cytosolic calcium ([Ca(2+)](cyt)) and stomatal closure in guard cells. ABA and ROS elicited [Ca(2+)](cyt) oscillations in guard cells only when extracellular Ca(2+) was present. In a 5 mM KCl extracellular buffer, 45% of guard cells exhibited spontaneous [Ca(2+)](cyt) oscillations that differed in their kinetic properties from ABA-induced Ca(2+) increases. These spontaneous [Ca(2+)](cyt) oscillations also required the availability of extracellular Ca(2+) and depended on the extracellular potassium concentration. Interestingly, when ABA was applied to spontaneously oscillating cells, ABA caused cessation of [Ca(2+)](cyt) elevations in 62 of 101 cells, revealing a new mode of ABA signaling. These data show that fungal elicitors activate a shared branch with ABA in the stress signal transduction pathway in guard cells that activates plasma membrane I(Ca) channels and support a requirement for extracellular Ca(2+) for elicitor and ABA signaling, as well as for cellular [Ca(2+)](cyt) oscillation maintenance.     

Both calcium and ROS as common signals mediate Na(2)SeO(3)-induced apoptosis in SW480 human colonic carcinoma cells

Recent studies have shown that reactive oxygen species (ROS) play a crucial role in Se-induced cell apoptosis. A number of studies have demonstrated that perturbed cellular calcium homeostasis has been implicated in apoptosis. The main objective of this study was to evaluate the role of Ca(2+) in Na(2)SeO(3)-induced apoptosis and the relationship between Ca(2+) and ROS in human colonic carcinoma cells SW480. When SW480 cells were exposed to 25-100 microM Na(2)SeO(3), both cell apoptosis and growth inhibition were observed by flow cytometric analysis and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Na(2)SeO(3) was able to induce increase of [Ca(2+)](i) and ROS production and disrupt mitochondrial membrane potential (Delta Psi m) in SW480 cells monitored by using a confocal laser scanning microscope. Ca(2+) channel inhibitor CoCl(2) and an intracellular Ca(2+) chelator o-phtalaldehyde, 1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetra-acetic acid acetoxymethyl ester (BAPTA) completely inhibited [Ca(2+)](i) increase, but catalase had no effect on Na(2)SeO(3)-induced increase of [Ca(2+)](i). BAPTA-AM, CoCl(2), and mitochondrial Ca(2+) uptake inhibitor ruthenium red blocked Delta Psi m dissipation. The increase of ROS was also suppressed by CoCl(2), BAPTA, ruthenium red, N-acetylcysteine and catalase, respectively. The mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) completely inhibited Na(2)SeO(3)-induced ROS increase. This showed that ROS increase is due to mitochondrial Ca(2+) overload. The Na(2)SeO(3)-induced apoptosis of SW480 cells was also inhibited by CoCl(2), BAPTA, ruthenium red, N-acetylcysteine, and catalase, respectively. The results mentioned above imply that both calcium and Ca(2+)-dependent ROS as a signal molecule mediate apoptosis induced by Na(2)SeO(3) in SW480 cells.     

Hypoxia-induced alterations in Ca(2+) mobilization in brain microvascular endothelial cells

To investigate the possible cellular mechanisms of the ischemia-induced impairments of cerebral microcirculation, we investigated the effects of hypoxia/reoxygenation on the intracellular Ca(2+) concentration ([Ca(2+)](i)) in bovine brain microvascular endothelial cells (BBEC). In the cells kept in normal air, ATP elicited Ca(2+) oscillations in a concentration-dependent manner. When the cells were exposed to hypoxia for 6 h and subsequent reoxygenation for 45 min, the basal level of [Ca(2+)](i) was increased from 32.4 to 63.3 nM, and ATP did not induce Ca(2+) oscillations. Hypoxia/reoxygenation also inhibited capacitative Ca(2+) entry (CCE), which was evoked by thapsigargin (Delta[Ca(2+)](i-CCE): control, 62.3 +/- 3.1 nM; hypoxia/reoxygenation, 17.0 +/- 1.8 nM). The impairments of Ca(2+) oscillations and CCE, but not basal [Ca(2+)](i), were restored by superoxide dismutase and the inhibitors of mitochondrial electron transport, rotenone and thenoyltrifluoroacetone (TTFA). By using a superoxide anion (O(2)(-))-sensitive luciferin derivative MCLA, we confirmed that the production of O(2)(-) was induced by hypoxia/reoxygenation and was prevented by rotenone and TTFA. These results indicate that hypoxia/reoxygenation generates O(2)(-) at mitochondria and impairs some Ca(2+) mobilizing properties in BBEC.     

Melatonin-induced calcium signaling in clusters of human and rat duodenal enterocytes

The amount of melatonin present in enterochromaffin cells in the alimentary tract is much higher than that in the central nervous system, and melatonin acting at MT(2) receptors mediates neural stimulation of mucosal HCO(3)(-) secretion in duodenum in vivo. We have examined effects of melatonin and receptor ligands on intracellular free calcium concentration ([Ca(2+)](i)) signaling in human and rat duodenal enterocytes. Clusters of interconnecting enterocytes (10-50 cells) were isolated by mild digestion (collagenase/dispase) of human duodenal biopsies or rat duodenal mucosa loaded with fura-2 AM and attached to the bottom of a temperature-controlled perfusion chamber. Clusters provided viable preparations and respond to stimuli as a syncytium. Melatonin and melatonin receptor agonists 2-iodo-N-butanoyl-5-methoxytryptamine and 2-iodomelatonin (1.0-100 nM) increased enterocyte [Ca(2+)](i), EC(50) of melatonin being 17.0 +/- 2.6 nM. The MT(2) receptor antagonists luzindole and N-pentanoyl-2-benzyltryptamine abolished the [Ca(2+)](i) responses. The muscarinic antagonist atropine (1.0 microM) was without effect on basal [Ca(2+)](i) and did not affect the response to melatonin. In the main type of response, [Ca(2+)](i) spiked rapidly and returned to basal values within 4-6 min. In another type, the initial rise in [Ca(2+)](i) was followed by rhythmic oscillations of high amplitude. Melatonin-induced enterocyte [Ca(2+)](i) signaling as well as mucosal cell-to-cell communication may be involved in stimulation of duodenal mucosal HCO(3)(-) secretion.     

Anion channel activation and proton pumping inhibition involved in the plasma membrane depolarization induced by ABA in Arabidopsis thaliana suspension cells are both ROS dependent

In Arabidopsis thaliana suspension cells, ABA was previously shown to promote the activation of anion channels and the reduction of proton pumping that both contribute to the plasma membrane depolarization. These two ABA responses were shown to induce two successive [Ca(2+)](cyt) spikes. As reactive oxygen species (ROS) have emerged as components of ABA signaling pathways especially by promoting [Ca(2+)](cyt) variations, we studied whether ROS were involved in the regulation of anion channels and proton pumps activities. Here we demonstrated that ABA induced ROS production which triggered the second of the two [Ca(2+)](cyt) increases observed in response to ABA. Blocking ROS generation using diphenyleneiodonium (DPI) impaired the proton pumping reduction, the anion channel activation and the RD29A gene expression in response to ABA. Furthermore, H(2)O(2) was shown to activate anion channels and to inhibit plasma membrane proton pumping, as did ABA. However, ROS partially mimicked ABA's effects since H(2)O(2) treatment elicited anion channel activation but not the subsequent expression of the RD29A gene as did ABA. This suggests that expression of the RD29A gene in response to ABA results from the activation of multiple concomitant signaling pathways: blocking of one of them would impair gene expression whereas stimulating only one would not. We conclude that ROS are a central messenger of ABA in the signaling pathways leading to the plasma membrane depolarization induced by ABA.     

Interleukin-8 primes oxidative burst in neutrophil-like HL-60 through changes in cytosolic calcium

In response to a variety of stimuli, neutrophils release large amount of reactive oxygen species (ROS) generated by NADPH oxidase. This process known as the respiratory burst is dependent on cytosolic free calcium concentration ([Ca(2+)](i)). Proinflammatory cytokines such as interleukin-8 (IL-8) may modulate ROS generation through a priming phenomenon. The aim of this study was to determine the effect of human IL-8 on ROS production in neutrophil-like dimethylsulfoxide-differentiated HL-60 cells (not equalHL-60 cells) and further to examine the role of Ca(2+) mobilization during the priming. IL-8 at 10 nM induced no ROS production but a [Ca(2+)](i) rise (254 +/- 36 nM). IL-8 induced a strongly enhanced (2 fold) ROS release during stimulation with 1 microM of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF). This potentiation of ROS production is dependent of extracellular Ca(2+) (17.0+/-4.5 arbitrary units (A.U.) in the absence of Ca(2+) versus 56.6 +/- 3.9 A.U. in the presence of 1.25 mM of Ca(2+)). Also, IL-8 enhanced fMLF-stimulated increase in [Ca(2+)](i) (375 +/- 35 versus 245 +/- 21 nM, 0.1 microM of fMLF). IL-8 had no effect on not equalHL-60 cells in response to 1 microM of thapsigargin (472 +/- 66 versus 470 +/- 60 nM). In conclusion, Ca(2+) influx is necessary for a full induction of neutrophil priming by IL-8.     

Novel effects of gossypol, a chemical contraceptive in man: mobilization of internal Ca(2+) and activation of external Ca(2+) entry in intact cells

The effect of gossypol on Ca(2+) signaling in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Gossypol evoked a rise in cytosolic free Ca(2+) levels ([Ca(2+)](i)) concentration-dependently between 2 and 20 microM. The response was decreased by external Ca(2+) removal. In Ca(2+)-free medium pretreatment with gossypol nearly abolished the [Ca(2+)](i) increase induced by carbonylcyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, and thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) pump; but pretreatment with CCCP and thapsigargin only partly inhibited gossypol-induced Ca(2+) release. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) increase after pretreatment with 5 microM gossypol in Ca(2+)-free medium. This Ca(2+) entry was decreased by 25 microM econazole, 50 microM SKF96365 and 40 microM aristolochic acid (a phospholipase A(2) inhibitor). Pretreatment with aristolochic acid inhibited 5 microM gossypol-induced internal Ca(2+) release by 55%, but suppression of phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) had no effect. Gossypol (5 microM) also increased [Ca(2+)](i) in human bladder cancer cells and neutrophils. Collectively, we have found that gossypol increased [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from multiple Ca(2+) stores in a manner independent of the production of inositol-1,4,5-trisphosphate, followed by Ca(2+) influx from external space.     

ANG II signaling in vasa recta pericytes by PKC and reactive oxygen species

ANG II constricts descending vasa recta (DVR) through Ca(2+) signaling in pericytes. We examined the role of PKC DVR pericytes isolated from the rat renal outer medulla. The PKC blocker staurosporine (10 microM) eliminated ANG II (10 nM)-induced vasoconstriction, inhibited pericyte cytoplasmic Ca(2+) concentration ([Ca(2+)](cyt)) elevation, and blocked Mn(2+) influx into the cytoplasm. Activation of PKC by either 1,2-dioctanoyl-sn-glycerol (10 microM) or phorbol 12,13-dibutyrate (PDBu; 1 microM) induced both vasoconstriction and pericyte [Ca(2+)](cyt) elevation. Diltiazem (10 microM) blocked the ability of PDBu to increase pericyte [Ca(2+)](cyt) and enhance Mn(2+) influx. Both ANG II- and PDBu-induced PKC stimulated DVR generation of reactive oxygen species (ROS), measured by oxidation of dihydroethidium (DHE). The effect of ANG II was only significant when ANG II AT(2) receptors were blocked with PD-123319 (10 nM). PDBu augmentation of DHE oxidation was blocked by either TEMPOL (1 mM) or diphenylene iodonium (10 microM). We conclude that ANG II and PKC activation increases DVR pericyte [Ca(2+)](cyt), divalent ion conductance into the cytoplasm, and ROS generation.     

Nitric oxide affects the production of reactive oxygen species in hepatoma cells: implications for the process of oxygen sensing

Treatment of human hepatoma cells (HepG2) with NO-donors for 24 h inhibited hypoxia-induced erythropoietin (EPO) gene activation. NO was found to increase the production of reactive oxygen species (ROS), the putative signaling molecules between a cellular O2-sensor and hypoxia inducible factor 1 (HIF-1). HIF-1 is the prime regulator of O2-dependent genes such as EPO. NO-treatment for more than 20 h reduced HIF-1-driven reporter gene activity. In contrast, immediately after the addition of NO, ROS levels in HepG2 cells decreased below control values for as long as 4 h. Corresponding to these lowered ROS-levels, HIF-1 reporter gene activity and EPO gene expression transiently increased but were reduced when ROS levels rose thereafter. Our findings of a bimodal effect of NO on ROS production shed new light on the involvement of ROS in the mechanism of O2-sensing and may explain earlier conflicting data about the effect of NO on O2-dependent gene expression.