Receptor-mediated effect of a synthetic thromboxane-analogue on cytosolic calcium in isolated proximal tubules.

The aims of this study were to measure cytosolic calcium concentration -[Ca2+]i- under resting conditions in isolated renal proximal tubules and to analyze the effect of U-46619 (stable analogue of thromboxane A2/PGH2 on [Ca2+]i in a mammalian epithelium. Proximal tubules were dissected out from male New Zealand rabbits (2.5 to 3.0 kg). After isolation they were washed twice and resuspended in 2 ml phosphate buffer solution (PBS). Tubules were loaded with Quin 2-AM (25 microM) for 15 min. After washing with PBS to eliminate the excess of extracellular Quin 2, fluorescence was measured at 340 nm excitation and 490 emission, under resting conditions and after stimulation. U 46619 (from 10 nm to 10 mM) increased [Ca2+]i in a concentration-dependent pattern. Exposure to an antagonist of the thromboxane receptor (S-145) blocked the response to U-46619. Removal of external calcium abolished the response to U-46619. Change of PBS for Ringer-choline blunted the response to thromboxane analogue. Our results indicate that U-46619 increases cytosolic calcium through a receptor-mediated mechanism that requires external calcium to operate. Blockade of the response in the absence of external sodium suggests that Na+/Ca2+ exchanger participates in this response.     

Early activation of p160ROCK by pressure overload in rat heart

We investigated the mechanisms underlying regulation of contraction with measurements of isometric force and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in NIH 3T3 fibroblast reconstituted into fibers with the use of a collagen matrix. Treatment with the major phospholipids, neurotransmitters, and growth factors had little effect on baseline isometric force. However, U-46619, a thromboxane A₂ (TxA₂) analog, increased force and [Ca²⁺]_i; EC₅₀ values were 11.0 and 10.0 nM, respectively. The time courses were similar to those induced by calf serum (CS), and the maximal force was 65% of a CS-mediated contraction. The selective TxA₂ receptor antagonist SQ-29548 abolished the U-46619-induced responses. CS-induced contractions are dependent on an intracellular Ca²⁺ store function; however, the U-46619 response depended not only on intracellular Ca²⁺ stores, but also on Ca²⁺ influx from the extracellular medium. Inhibition of Rho kinase suppressed U-46619- and CS-induced responses; in contrast, inhibition of C kinase (PKC) reduced only the U-46619 response. Moreover, addition of U-46619 to a CS contracture enhanced force and [Ca²⁺]_i responses. These results indicate that U-46619-induced responses involve PKC and Rho kinase pathways, in contrast to activation by CS. Thus TxA₂ may have a role in not only the initial step of wound repair as an activator of blood coagulation, but also in fibroblast contractility in later stages. collagen matrix; signal transduction; wound repair     

Thromboxane A2 stimulates mitogen activated protein kinase and arachidonic acid liberation in rabbit platelets

U46619, a thromboxane A₂ mimetic, caused tyrosine phosphorylation of several proteins in rabbit platelets. Among them, 42 kDa protein was identified as a mitogen-activated protein kinase (MAPK). U46619 activated MAPK in a concentration-dependent manner, measured by incorporation of ³²P to a specific substrate for MAPK. U46619 also liberated [³H)arachidonic acid in a concentration-dependent manner. The U46619-induced MAPK activation and [³H]arachidonic acid liberation were inhibited by SQ29548 and by the removal of external Ca²⁺ ions. This is a first demonstration that TXA₂ activates MAPK accompanied with arachidonic acid liberation in rabbit platelets.     

Cyclic Nucleotide-Gated Channels Contribute to Thromboxane A2-Induced Contraction of Rat Small Mesenteric Arteries

Background

Thromboxane A₂ (TxA₂)-induced smooth muscle contraction has been implicated in cardiovascular, renal and respiratory diseases. This contraction can be partly attributed to TxA₂-induced Ca²⁺ influx, which resulted in vascular contraction via Ca²⁺-calmodulin-MLCK pathway. This study aims to identify the channels that mediate TxA₂-induced Ca²⁺ influx in vascular smooth muscle cells.

Methodology/Principal Findings

Application of U-46619, a thromboxane A₂ mimic, resulted in a constriction in endothelium-denuded small mesenteric artery segments. The constriction relies on the presence of extracellular Ca²⁺, because removal of extracellular Ca²⁺ abolished the constriction. This constriction was partially inhibited by an L-type Ca²⁺ channel inhibitor nifedipine (0.5–1 µM). The remaining component was inhibited by L-cis-diltiazem, a selective inhibitor for CNG channels, in a dose-dependent manner. Another CNG channel blocker LY83583 [6-(phenylamino)-5,8-quinolinedione] had similar effect. In the primary cultured smooth muscle cells derived from rat aorta, application of U46619 (100 nM) induced a rise in cytosolic Ca²⁺ ([Ca²⁺]_i), which was inhibited by L-cis-diltiazem. Immunoblot experiments confirmed the presence of CNGA2 protein in vascular smooth muscle cells.

Conclusions/Significance

These data suggest a functional role of CNG channels in U-46619-induced Ca²⁺ influx and contraction of smooth muscle cells.     

Stimulation of cytosolic free calcium and inositol phosphates by prostaglandins in cultured rat mesangial cells

We studied the effects of four products of arachidonate cyclo-oxygenation on a phospholipase C-dependent signal transduction system in cultured rat glomerular mesangial cells. PGF2 alpha, PGE2 and the thromboxane A2/endoperoxide analogue U-46619 rapidly increased cytosolic free Ca2+, measured in monolayers loaded with the fluorescent intracellular probe fura-2. Peak responses were dose-dependent and unaffected by chelation of extracellular Ca2+, indicating release from internal stores. The thromboxane A2-receptor antagonist SQ 27,427 selectively inhibited responses to U-46619. The PGI2 analogue Iloprost had no effect on cytosolic Ca2+. PGF2 alpha, PGE2 and U-46619 also stimulated accumulation of total inositol phosphates during 15 min incubations. We conclude that phospholipase C activation mediates the effects of certain eicosanoids on the glomerular mesangium.     

Relationship between calcium release and NADPH oxidase inhibition in human neutrophils

The aim of this study was to investigate the possible relationship between NADPH oxidase activity and changes in cytosolic Ca²⁺ in response to different agonists. Treatment of neutrophils with leukotriene B4 (LTB₄) demonstrated characteristic changes to cytoslic Ca²⁺ yielding an EC₅₀ of 4 nM. The pA₂ values for the specific LTB₄ receptor (BLT) antagonists, U-75302 and LY-255283 were 6.32 and 6.38, respectively. Similarly, neutrophils treated with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and platelet activating factor (PAF) exhibited changes in cytoslic Ca²⁺ in a dose dependant manner with pD₂ values of 9.0 and 9.9, respectively. The phorbol ester PMA prevented elevations in cytosolic Ca²⁺ in response to LTB₄, FMLP and PAF with IC₅₀ values of 5.88, 1.44 and 5.71 nM, respectively. In addition, potent NADPH oxidase inhibitors apocynin and diphenyleneiodonium (DPI) inhibited FMLP mediated cytosolic Ca²⁺ release. These results demonstrate that inhibition of the NADPH oxidase suppresses cytosolic Ca²⁺ release in FMLP activated human neutrophils.     

Oxidative stress disruption of receptor-mediated calcium signaling mechanisms

Background

Oxidative stress increases the cytosolic content of calcium in the cytoplasm through a combination of effects on calcium pumps, exchangers, channels and binding proteins. In this study, oxidative stress was produced by exposure to tert-butyl hydroperoxide (tBHP); cell viability was assessed using a dye reduction assay; receptor binding was characterized using [³H]N-methylscopolamine ([³H]MS); and cytosolic and luminal endoplasmic reticulum (ER) calcium concentrations ([Ca²⁺]_i and [Ca²⁺]_L, respectively) were measured by fluorescent imaging.

Results

Activation of M3 muscarinic receptors induced a biphasic increase in [Ca²⁺]_i: an initial, inositol trisphosphate (IP3)-mediated release of Ca²⁺ from endoplasmic reticulum (ER) stores followed by a sustained phase of Ca²⁺ entry (i.e., store-operated calcium entry; SOCE). Under non-cytotoxic conditions, tBHP increased resting [Ca²⁺]_i; a 90 minute exposure to tBHP (0.5-10 mM ) increased [Ca²⁺]_i from 26 to up to 127 nM and decreased [Ca²⁺]_L by 55%. The initial response to 10 μM carbamylcholine was depressed by tBHP in the absence, but not the presence, of extracellular calcium. SOCE, however, was depressed in both the presence and absence of extracellular calcium. Acute exposure to tBHP did not block calcium influx through open SOCE channels. Activation of SOCE following thapsigargin-induced depletion of ER calcium was depressed by tBHP exposure. In calcium-free media, tBHP depressed both SOCE and the extent of thapsigargin-induced release of Ca²⁺ from the ER. M3 receptor binding parameters (ligand affinity, guanine nucleotide sensitivity, allosteric modulation) were not affected by exposure to tBHP.

Conclusions

Oxidative stress induced by tBHP affected several aspects of M3 receptor signaling pathway in CHO cells, including resting [Ca²⁺]_i, [Ca²⁺]_L, IP3 receptor mediated release of calcium from the ER, and calcium entry through the SOCE. tBHP had little effect on M3 receptor binding or G protein coupling. Thus, oxidative stress affects multiple aspects of calcium homeostasis and calcium dependent signaling.     

Hydrocortisone inhibits antigen-induced rise in intracellular free calcium concentration and abolishes leukotriene C4 production in leukemic basophils

Antigenic stimulation of rat basophilic leukemia cells (RBL-3H3) elevates intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and induces production of leukotriene C₄ (LTC₄). This model was used to examine the role of Ca²⁺ in LTC₄ formation, and inhibition by hydrocortisone (HC). HC, at a physiological concentration (2×10⁻⁷M), selectively prevented the stimulatory effect of the antigen on LTC₄ production whereas the response to calcium inophore (A23187) remained unimpaired. The inhibition by HC was time-dependent: half maximal response was reached at 2 hour and maximal response at 3 hours. Addition of arachidonic acid (3 μg/ml) did not overcome the inhibitory action of HC. An elevated [Ca²⁺]_i is known to be essential for the activation ob both 5-lipoxygenase and phospholipase A₂. The stimulatory effect of the antigen on LTC₄ production was abolished when the cells were incubated in Ca²⁺-deficient medium. Likewise, calcium ionophore stimulation shows dependence on extracellular Ca²⁺. Half maximal stimulation by the antigen and calcium ionophore was observed at external Ca²⁺ concentration of 150 μM and 40 μM respectively. Treatment with HC largely prevented the antigen-induced rise in [Ca²⁺]_i, measured by Quin 2. In addition, HC reduced by 70% the accumulation of ⁴⁵Ca²⁺ induced by the antigen. Collectively, these results demonstrate for the first time that HC reduces antigen-induced elevation of [Ca²⁺]_i, and this may be associated with the inhibitory action of HC on LTC₄ formation. This property could be partly responsible for the antiallergic and antiinflammatory activities of HC.     

Effects of differentiation on purinergic and neurotensin-mediated calcium signaling in human HT-29 colon cancer cells

Calcium signaling is a key regulator of processes important in differentiation. In colon cancer cells differentiation is associated with altered expression of specific isoforms of calcium pumps of the endoplasmic reticulum and the plasma membrane, suggesting that differentiation of colon cancer cells is associated with a major remodeling of calcium homeostasis. Purinergic and neurotensin receptor activation are known regulators of cytosolic free Ca²⁺ levels in colon cancer cells. This study aimed to assess changes in cytosolic free Ca²⁺ levels in response to ATP and neurotensin with differentiation induced by sodium butyrate or culturing post-confluence. Parameters assessed included peak cytosolic free Ca²⁺ level after activation; time to reach peak cytosolic free Ca²⁺ and the EC₅₀ of dose response curves. Our results demonstrate that differentiation of HT-29 colon cancer cells is associated with a remodeling of both ATP and neurotensin mediated Ca²⁺ signaling. Neurotensin-mediated calcium signaling appeared more sensitive to differentiation than ATP-mediated Ca²⁺ signaling.     

Cytosolic free calcium concentrations in synaptosomes during histotoxic hypoxia

Altered cytosolic free calcium concentrations ([Ca²⁺]_i) accompany impaired brain metabolism and may mediate subsequent effects on brain function and cell death. The current experiments examined whether hypoxia-induced elevations in [Ca²⁺]_i are from external or internal sources. In the absence of external calcium, neither KCl depolarization, histotoxic hypoxia (KCN), nor the combination changed [Ca²⁺]_i. However, with external CaCl₂ concentrations as small as 13 M, KCl depolarization increased [Ca²⁺]_i instantaneously while hypoxia gradually raised [Ca²⁺]_i. The combination of KCN and KCl was additive. Increasing external calcium concentrations up to 2.6 mM exaggerated the effects of K⁺ and KCN on [Ca²⁺]_i, but raising medium calcium to 5.2 mM did not further augment the rise. Diminishing the sodium in the media, which alters the activity and perhaps the direction of the Na/Ca exchanger, reduced the increase in [Ca²⁺]_i due to hypoxia, but enhanced the KCl response. The changes in ATP following K⁺ depolarization, KCN or their combination in the presence of physiological calcium concentrations did not parallel alterations in [Ca²⁺]_i, which suggests that diminished activity of the calcium dependent ATPase does not underlie the elevation in [Ca²⁺]_i. Valinomycin, an ionophore which reduces the mitochondrial membrane potential, elevated [Ca²⁺]_i and the effects were additive with K⁺ depolariration in a calcium dependent manner that paralleled the effects of hypoxia. Together these results suggest that hypoxia-induced elevations of synaptosomal [Ca²]_i are due to an inability of the synaptosome to buffer entering calcium.     

Measurements of intracellular ionized calcium in squid giant axons using calcium-selective electrodes

Ca²⁺-selective electrodes have been used to measure free intracellular Ca²⁺ concentrations in squid giant axons. Electrodes made of glass cannulas of about 20 μm in diameter, plugged with a poly(vinyl chloride) gelled sensor were used to impale the axons axially. They showed a Nernstian response to Ca²⁺ down to about 3 μM in solutions containing 0.3 M K⁺ and 0.025 M Na⁺. Sub-Nernstian but useful responses were obtained up to pCa 8. The electrodes showed adequate selectivity to Ca²⁺ over Mg²⁺, H⁺, K⁺ and Na⁺. To calibrate them properly, a set of standard solutions were prepared using different Ca²⁺ buffers (EGTA, HEEDTA, nitrilotriacetic acid) after carefully characterizing their apparent Ca²⁺ association constants under conditions resembling the axoplasmic environment. In fresh axons incubated in artificial seawater containing 4 mM Ca²⁺, the mean resting intracellular ionized calcium concentration was 0.106 μM ($\text{n = 15}$). The Ca²⁺-electrodes were used to investigate effects of different experimental procedures on the [Ca²⁺]_i. The main conclusions are: (i) intact axons can extrude calcium ions at low [Ca²⁺]_i levels by a process independent of external Na⁺; (ii) poisoned axons can extrude calcium ions at high levels of [Ca²⁺]_i by an external Na⁺-dependent process. The level of free intracellular Ca attained at these latter conditions is about an order to magnitude greater than the resting physiological value.     

Rho activation in excitatory agonist-stimulated vascular smooth muscle

Small GTPase Rho and its downstream effector, Rho kinase, havebeen implicated in agonist-stimulated Ca²⁺ sensitization of20-kDa myosin light chain (MLC₂₀) phosphorylation andcontraction in smooth muscle. In the present study we demonstrated forthe first time that excitatory receptor agonists induce increases inamounts of an active GTP-bound form of RhoA, GTP-RhoA, in rabbit aorticsmooth muscle. Using a pull-down assay with a recombinant RhoA-bindingprotein, Rhotekin, we found that a thromboxane A₂ mimetic,U-46619, which induced a sustained contractile response, induced asustained rise in the amount of GTP-RhoA in a dose-dependent mannerwith an EC₅₀ value similar to that for the contractile response. U-46619-induced RhoA activation was thromboxaneA₂ receptor-mediated and reversible. Other agonistsincluding norepinephrine, serotonin, histamine, and endothelin-1 (ET-1)also stimulated RhoA, albeit to lesser extents than U-46619. Incontrast, ANG II and phorbol 12,13-dibutyrate failed to increaseGTP-RhoA. The tyrosine kinase inhibitor genistein substantiallyinhibited RhoA activation by these agonists, except for ET-1. Thusexcitatory agonists induce Rho activation in an agonist-specificmanner, which is thought to contribute to stimulation ofMLC₂₀ phosphorylation Ca²⁺ sensitivity.

     

Mitochondrial Ca2+-Handling in Fast Skeletal Muscle Fibers from Wild Type and Calsequestrin-Null Mice

Mitochondrial calcium handling and its relation with calcium released from sarcoplasmic reticulum (SR) in muscle tissue are subject of lively debate. In this study we aimed to clarify how the SR determines mitochondrial calcium handling using dCASQ-null mice which lack both isoforms of the major Ca²⁺-binding protein inside SR, calsequestrin. Mitochondrial free Ca²⁺-concentration ([Ca²⁺]_mito) was determined by means of a genetically targeted ratiometric FRET-based probe. Electron microscopy revealed a highly significant increase in intermyofibrillar mitochondria (+55%) and augmented coupling (+12%) between Ca²⁺ release units of the SR and mitochondria in dCASQ-null vs. WT fibers. Significant differences in the baseline [Ca²⁺]_mito were observed between quiescent WT and dCASQ-null fibers, but not in the resting cytosolic Ca²⁺ concentration. The rise in [Ca²⁺]_mito during electrical stimulation occurred in 20−30 ms, while the decline during and after stimulation was governed by 4 rate constants of approximately 40, 1.6, 0.2 and 0.03 s⁻¹. Accordingly, frequency-dependent increase in [Ca²⁺]_mito occurred during sustained contractions. In dCASQ-null fibers the increases in [Ca²⁺]_mito were less pronounced than in WT fibers and even lower when extracellular calcium was removed. The amplitude and duration of [Ca²⁺]_mito transients were increased by inhibition of mitochondrial Na⁺/Ca²⁺ exchanger (mNCX). These results provide direct evidence for fast Ca²⁺ accumulation inside the mitochondria, involvement of the mNCX in mitochondrial Ca²⁺-handling and a dependence of mitochondrial Ca²⁺-handling on intracellular (SR) and external Ca²⁺ stores in fast skeletal muscle fibers. dCASQ-null mice represent a model for malignant hyperthermia. The differences in structure and in mitochondrial function observed relative to WT may represent compensatory mechanisms for the disease-related reduction of calcium storage capacity of the SR and/or SR Ca²⁺-leakage.     

Resting potential of rat cerebellar granule cells during early maturation in vitro

The survival of rat cerebellar granule cells maintained in vitro is enhanced by a KCl-enriched medium. This effect is classically interpreted as resulting from a higher cytosolic calcium concentration. This implies the presence of voltage-dependent Ca²⁺ channels and a membrane potential that can respond to changes in external K⁺. Since previous studies cast a doubt on these two conditions, we reinvestigated the resting membrane potential and Ca²⁺ influxes in rat cerebellar granule neurones during the first week in vitro using a fluorescence imaging approach. Membrane potential was assessed with the fluorescent dye bis-oxonol, and intracellular free calcium with Fura-2. Resting potential was shown to progressively decrease from −40 mV at the first day in vitro to −60 mV at day 7. At all times in culture, as early as day 0, cells were depolarized when external KCl concentration was increased from 5 to 30 mM. This depolarization resulted in an increased cytosolic calcium concentration due to Ca²⁺ influx through L-type and N-type voltage-activated Ca²⁺ channels, functional at day 0. Gross estimations of the permeabilities of Na⁺ and Cl⁻ were obtained at various times in culture by measuring the changes in resting potential brought about by a reduction of their external concentration. A progressive increase of the relative permeability to K⁺ ions seems to underlie the evolution of the resting potential with time. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 11–21, 1997.     

Hypoxia induces hypersensitivity and hyperreactivity to thromboxane receptor agonist in neonatal pulmonary arterial myocytes

PPHN, caused by perinatal hypoxia or inflammation, is characterized by an increased thromboxane-prostacyclin ratio and pulmonary vasoconstriction. We examined effects of hypoxia on myocyte thromboxane responsiveness. Myocytes from 3rd-6th generation pulmonary arteries of newborn piglets were grown to confluence and synchronized in contractile phenotype by serum deprivation. On the final 3 days of culture, myocytes were exposed to 10% O2 for 3 days; control myocytes from normoxic piglets were cultured in 21% O2. PPHN was induced in newborn piglets by 3-day hypoxic exposure (Fi(O2) 0.10); pulmonary arterial myocytes from these animals were maintained in normoxia. Ca2+ mobilization to thromboxane mimetic U-46619 and ATP was quantified using fura-2 AM. Three-day hypoxic exposure in vitro results in increased basal [Ca2+]i, faster and heightened peak Ca2+ response, and decreased U-46619 EC50. These functional changes persist in myocytes exposed to hypoxia in vivo but cultured in 21% O2. Blockade of Ca2+ entry and store refilling do not alter peak U-46619 Ca2+ responses in hypoxic or normoxic myocytes. Blockade of ryanodine-sensitive or IP3-gated intracellular Ca2+ channels inhibits hypoxic augmentation of peak U-46619 response. Ca2+ response to ryanodine alone is undetectable; ATP-induced Ca2+ mobilization is unaltered by hypoxia, suggesting no independent increase in ryanodine-sensitive or IP3-linked intracellular Ca2+ pool mobilization. We conclude hypoxia has a priming effect on neonatal pulmonary arterial myocytes, resulting in increased resting Ca2+, thromboxane hypersensitivity, and hyperreactivity. We postulate that hypoxia increases agonist-induced TP-R-linked IP3 pathway activation. Myocyte thromboxane hyperresponsiveness persists in culture after removal from the initiating hypoxic stimulus, suggesting altered gene expression.     

Calcium and ATP handling in human NADH:Ubiquinone oxidoreductase deficiency

Proper cell functioning requires precise coordination between mitochondrial ATP production and local energy demand. Ionic calcium (Ca²⁺) plays a central role in this coupling because it activates mitochondrial oxidative phosphorylation (OXPHOS) during hormonal and electrical cell stimulation. To determine how mitochondrial dysfunction affects cytosolic and mitochondrial Ca²⁺/ATP handling, we performed life-cell quantification of these parameters in fibroblast cell lines derived from healthy subjects and patients with isolated deficiency of the first OXPHOS complex (CI). In resting patient cells, CI deficiency was associated with a normal mitochondrial ([ATP]_m) and cytosolic ([ATP]_c) ATP concentration, a normal cytosolic Ca²⁺ concentration ([Ca²⁺]_c), but a reduced Ca²⁺ content of the endoplasmic reticulum (ER). Furthermore, cellular NAD(P)H levels were increased, mitochondrial membrane potential was slightly depolarized, reactive oxygen species (ROS) levels were elevated and mitochondrial shape was altered. Upon stimulation with bradykinin (Bk), the peak increases in [Ca²⁺]_c, mitochondrial Ca²⁺ concentration ([Ca²⁺]_m), [ATP]_c and [ATP]_m were reduced in patient cells. In agreement with these results, ATP-dependent Ca²⁺ removal from the cytosol was slower. Here, we review the interconnection between cytosolic, endoplasmic reticular and mitochondrial Ca²⁺ and ATP handling, and summarize our findings in patient fibroblasts in an integrative model.     

Implication of the nucleus in excitation contraction coupling of heart cells

In the present study, Fluo-3 Ca²⁺ measurement and confocal microscopy techniques were used in order to localize cytosolic [ ]_c and nuclear [ ]_n free Ca²⁺ distribution in resting and spontaneously contracting single heart cells from 10-day-old chick embryos. In resting single cells, the concentration of Ca²⁺ in the cytoplasm was lower than that in the nucleus. Increasing cytosolic free Ca²⁺ from 100–1600 nM gradually increased [Ca²⁺]_n with a maximum capacity near 1200 nM. Results from Fura-2 microfluorometry and Fluo-3 confocal microscopy suggest a potential cross talk between the increase of cytosolic free Ca²⁺ and the uptake and release of Ca²⁺ by the nucleus during spontaneous contraction of single myocytes. Calcium waves in spontaneously contracting cells were found to spread from one cell to the next with the nucleus acting as a fluorescent beacon in which Ca²⁺ levels remained elevated for several milliseconds even after cytosolic Ca²⁺ had returned to near basal values. These results strongly suggest that the nucleus plays a negative and positive feedback role in controlling cytosolic free Ca²⁺ concentration during excitation-contraction coupling in heart cells.     

Sulfhydryl modification induces calcium entry through IP₃-sensitive store-operated pathway in activation-dependent human neutrophils

As the first line of host defense, neutrophils are stimulated by pro-inflammatory cytokines from resting state, facilitating the execution of immunomodulatory functions in activation state. Sulfhydryl modification has a regulatory role in a wide variety of physiological functions through mediation of signaling transductions in various cell types. Recent research suggested that two kinds of sulfhydryl modification, S-nitrosylation by exogenous nitric oxide (NO) and alkylation by N-ethylmaleimide (NEM), could induce calcium entry through a non-store-operated pathway in resting rat neutrophils and DDT₁MF-2 cells, while in active human neutrophils a different process has been observed by us. In the present work, data showed that NEM induced a sharp rising of cytosolic calcium concentration ([Ca²⁺]_c) without external calcium, followed by a second [Ca²⁺]_c increase with readdition of external calcium in phorbol 12-myristate 13-acetate (PMA)-activated human neutrophils. Meanwhile, addition of external calcium did not cause [Ca²⁺]_c change of Ca²⁺-free PMA-activated neutrophils before application of NEM. These data indicated that NEM could induce believable store-operated calcium entry (SOCE) in PMA-activated neutrophils. Besides, we found that sodium nitroprusside (SNP), a donor of exogenous NO, resulted in believable SOCE in PMA-activated human neutrophils via S-nitrosylation modification. In contrast, NEM and SNP have no effect on [Ca²⁺]_c of resting neutrophils which were performed in suspension. Furthermore, 2-Aminoethoxydiphenyl borate, a reliable blocker of SOCE and an inhibitor of inositol 1,4,5-trisphosphate (IP₃) receptor, evidently abolished SNP and NEM-induced calcium entry at 75 µM, while preventing calcium release in a concentration-dependent manner. Considered together, these results demonstrated that NEM and SNP induced calcium entry through an IP₃-sensitive store-operated pathway of human neutrophils via sulfhydryl modification in a PMA-induced activation-dependent manner.     

IP3-mediated cytosolic and nuclear calcium elevation in NRK-52E cells using ‘caged’ GPIP2

Alterations in calcium homeostasis play a pivotal role in the cellular response to injury. Increases in the concentration of cytosolic free calcium ([Ca²⁺]i) result in a variety of calcium mediated toxic responses such as cytoskeletal alterations, mitochondrial damage, and over-expression of gene products. Inositol trisphosphate is a second messenger that links external cell surface signals to [Ca²⁺]i elevation. The present study explored the use of caged glycerophosphoryl-myo-inositol-1,4,5-bisphosphate (GPIP₂) to mediate a rapid and prolonged increase in [Ca²⁺]i in a normal rat kidney epithelial cell line (NRK-52E). In intact NRK-52E cells, UV photolysis of microinjected GPIP₂ resulted in a 3–4-fold sustained increase in [Ca²⁺]_i. Graded photolytic release of GPIP2 also resulted in calcium-mediated morphologic alterations, as shown by confocal microscopy, with cellular blebs apparent within 30 min. There was no apparent increase in [Ca²⁺]i or morphologic alterations in control cells microinjected with calcium indicator and equally exposed to UV light. Subsequent application of thapsigargin or ionomycin (1.0 μM) produced a rapid and transient increase in [Ca²⁺]i. In addition, we show that activation of IN stores results in increased concentration of ionized nuclear calcium, ([Ca²⁺]n) which persists longer than the increase in [Ca²⁺]i. These findings indicate that GPIP₂ mediates a rapid and sustained elevation in [Ca²⁺]n and [Ca²⁺]i and this IP₃-mediated calcium elevation is translated to the nucleus in rat kidney epithelial cells.     

H2O2 Mobilizes Ca2+ from Agonist- and Thapsigargin-sensitive and Insensitive Intracellular Stores and Stimulates Glutamate Secretion in Rat Hippocampal Astrocytes

The effect of hydrogen peroxide (H₂O₂) on cytosolic free calcium concentration ([Ca²⁺]_c) as well as its effect on glutamate secretion in rat hippocampal astrocytes have been the aim of the present research. Our results show that 100 μM H₂O₂ induces an increase in [Ca²⁺]_c, that remains at an elevated level while the oxidant is present in the perfusion medium, due to its release from intracellular stores as it was observed in the absence of extracellular Ca²⁺, followed by a significant increase in glutamate secretion. Ca²⁺-mobilization in response to the oxidant could only be reduced by thapsigargin plus FCCP, indicating that the Ca²⁺-mobilizable pool by H₂O₂ includes both endoplasmic reticulum and mitochondria. We conclude that ROS in hippocampal astrocytes might contribute to an elevation of resting [Ca²⁺]_c which, in turn, could lead to a maintained secretion of the excitatory neurotransmitter glutamate, which has been considered a situation potentially leading to neurotoxicity in the hippocampus.