Prostaglandin E2 is a Potential Mediator of Extracellular ATP Action in Osteoblast-Like Cells

Extracellular ATP dose dependently stimulated ⁴⁵Ca²⁺ influx even in the presence of nifedipine, a Ca²⁺ antagonist that inhibits voltage-dependent Ca²⁺ channel, in osteoblast-like MC3T3-E1 cells. ATP stimulated arachidonic acid release and the synthesis of prostaglandin E₂ (PGE₂). However, the ATP-induced arachidonic acid release was significantly reduced by chelating extracellular Ca²⁺ with EGTA. On the other hand, ATP induced DNA synthesis of these cells in a dose-dependent manner in the range between 1μM and 1 mM. The pretreatment with indomethacin, a cyclooxygenase inhibitor, suppressed both ATP-induced PGE₂ synthesis and DNA synthesis in these cells. The inhibitory effect by 50μM indomethacin on the DNA synthesis was reversed by adding 10μM PGE₂. These results strongly suggest that extracellular ATP stimulates Ca²⁺ influx resulting in the release of arachidonic acid in osteoblast-like cells and that extracellular ATP-induced proliferative effect is mediated, at least in part, by ATP-stimulated PGE₂ synthesis.     

Metabolic acidosis,prostaglandin E2 and insulin stimulates intracellular free calcium ([Ca2+]1) in isolated cells of toad urinary bladder

It is well known that metabolic acidosis (MA), PGE₂, and insulin stimulate H⁺ excretion in toad urinary bladder. In addition, PGE₂ has been shown to increase in the toad bladder during MA. Our present experimental findings indicate that MA, PGE₂ and insulin increase [Ca²⁺]_i and this then may be the signal for stimulation of H⁺ excretion in this tissue. Isolated cells of the toad urinary bladder, obtained from toads in a chronic metabolic acidosis (MA) have a significantly higher intracellular Ca²⁺ ([Ca²⁺]_i) than similar cells obtained from toads in normal acid-base balance. Protaglandin E₂ (PGE₂) (10⁻⁵M) was found to stimulate [Ca²⁺]_i, in the same normal toad bladder cells, as determined by the fluorescence ratio technique using FURA 2/AM (P < 0.05). Insulin (100 mU/ml) was also found to stimulate [Ca²⁺]_i, in toad bladder cells (P < 0.01). The increase in [Ca²⁺]_i following PGE₂ stimulation was not dependent on extracellular Ca²⁺, whereas the increase seen following insulin stimulation was dependent on extracellular Ca²⁺.     

Thrombin, collagen and A23187 stimulated endogenous platelet arachidonate metabolism: Differential inhibition by PGE1, local anesthetics and a serine-protease inhibitor

The formation of malondialdehyde (MDA) and rabbit aorta contracting substance (RCS) induced by treatment of platelets with thrombin and collagen, but not that produced from exogenous arachidonic acid, is inhibited by prostaglandin E₁ (10⁻⁸ − 10⁻⁷M), the local anesthetics tetracaine, SKF 525-A and dibucaine (1 mM), and the serine-protease inhibitor phenylmethanesulfonyl fluoride (PMSF). The burst in oxygen consumption which accompanies platelet stimulation by thrombin and collagen in the presence of antimycin A, known to be due to the oxidation of endogenous arachidonate, is also markedly suppressed by PGE₁, tetracaine and PMSF. The inhibitory effect of PGE₁ is strongly potentiated by theophylline (1.0 mM).Addition of the Ca²⁺ ionophore A23187 to platelet suspensions overcomes PGE₁ and PMSF inhibition of MDA and RCS formation, and induces a vigorous increase in O₂ consumption. Tetracaine and dibucaine, however, block the responses to A23187.Formation of MDA and RCS (a mixture of PG endoperoxides and TXA₂) due to stimulation by thrombin and collagen depends upon activation of Ca²⁺-dependent phospholipase A₂ (PLA₂) to supply free arachidonate from specific membrane phospholipids. These experiments therefore indicate that increased cellular cAMP, induced by PGE₁, antagonizes the mobilization of the Ca²⁺ which is normally required for PLA₂ activity. Thrombin-stimulated platelets exhibit enhanced ⁴⁵Ca uptake which probably reflects exchange of extracellular Ca²⁺ with an increased available pool of exchangeable intracellular Ca²⁺. PGE₁ strongly suppresses this ⁴⁵Ca uptake, providing more direct evidence supporting the view that cAMP prevents the rise in free cytoplasmic Ca²⁺ induced by thrombin. Under conditions which make sufficient free cytoplasmic Ca²⁺ available (i.e., A23187), despite high cellular cAMP, formation of RCS and MDA, and O₂ uptake are nearly normal indicating that activation of PLA₂ can occur. Local anesthetics on the other hand since they abolish the response to A23187 as well, appear to directly antagonize the ability of Ca²⁺ to activate PLA₂. The effect of PMSF suggests that stimulus-specific proteases may be involved in the thrombin and collagen-induced activation of PLA₂ activity.     

Thrombin induces proliferation of osteoblast-like cells through phosphatidylcholine hydrolysis

We examined the effect of thrombin on phosphatidylcholine-hydrolyzing phospholipase D activity in osteoblast-like MC3T3-E1 cells. Thrombin stimulated the formation of choline dose dependently in the range between 0.01 and 1 U/ml, but not the phosphocholine formation. Diisopropylfluorophosphate (DFP)-inactivated thrombin had little effect on the choline formation. The combined effects of thrombin and 12-O-tetradecanoylphorbol-13-acetate, a protein kinase C-activating phorbol ester, on the choline formation were additive. Staurosporine, an inhibitor of protein kinases, had little effect on the thrombin-induced formation of choline. Combined addition of thrombin and NaF, an activator of heterotrimeric GTP-binding protein, did not stimulate the formation of choline further. Pertussis toxin had little effect on the thrombin-induced formation of choline. Thrombin stimulated Ca²⁺ influx from extracellular space time and dose dependently. The depletion of extracellular Ca²⁺ by EGTA exclusively reduced the thrombin-induced choline formation. Thrombin had only a slight effect on phosphoinositide-hydrolyzing phospholipase C activity. Thrombin induced diacylglycerol formation and DNA synthesis, and increased the number of MC3T3-E1 cells, but DFP-inactivated thrombin did not. Thrombin suppressed both basal and fetal calf serum-induced alkaline phosphatase activity in these cells. Propranolol, an inhibitor of phosphatidic acid phosphohydrolase, inhibited both the thrombin-induced diacylglycerol formation and DNA synthesis. These results suggest that thrombin stimulates phosphatidylcholine-hydrolyzing phospholipase D due to self-induced Ca²⁺ influx independently of protein kinase C activation in osteoblast-like cells and that its proliferative effect depends on phospholipase D activation. © 1996 Wiley-Liss, Inc.     

Selective Inhibition of the Expression of Signal Transduction Proteins by Lithium in Nerve Growth Factor-Differentiated PC12 Cells

Abstract: Muscarinic receptor in human neuroblastoma SK-N-BE(2)C cells was identified and characterized. Treatment of the cells with carbachol evoked the generation of inositol 1,4,5-trisphosphate (IP₃) with a peak level reached at 1 min after stimulation. Carbachol increased intracellular Ca²⁺ ([Ca²⁺]_i) with an EC₅₀ value of 35 µM. In addition, carbachol produced a 1.3–3-fold increase in the cyclic AMP (cAMP) level compared with untreated control and elevated synergistically the cAMP level in the treatment with prostaglandin E₂ (PGE₂). The M₃ antagonist p-fluorohexahydrosiladifenidol (IC₅₀ = 0.5–0.8 µM) inhibited the increases in [Ca²⁺]_i, IP₃, and cAMP more effectively than the M₁ antagonist pirenzepine (IC₅₀ = 5–9 µM) and the M₂ antagonist methoctramine (IC₅₀ = 20–30 µM). The involvements of [Ca²⁺]_i elevation and protein kinase C activation induced by phospholipase C activation were tested in the carbachol-induced cAMP production. The calcium chelator BAPTA/AM (75 µM) inhibited significantly the synergistic effects of carbachol and PGE₂ on the production of cAMP, whereas the Ca²⁺ ionophore ionomycin (1 µM) clearly enhanced PGE₂-induced cAMP production. However, phorbol 12-myristate 13-acetate did not enhance PGE₂-stimulated cAMP production. These data suggest that phospholipase C-linked M₃ receptors are present and that stimulation of the receptors activates adenylyl cyclase, at least in part, by the Ca²⁺-dependent system in the neuronal cells.     

Thrombin and histamine activate phospholipase C in human endothelial cells via a phorbol ester-sensitive pathway

The effects of phorbol esters and synthetic diglycerides on thrombin- and histamine-stimulated increases in inositol trisphosphate (IP₃) and cytosolic free calcium ([Ca²⁺]_i) were studied in cultured human umbilical vein endothelial cells (HEC). Thrombin (0.003–3.0 U/ml) and histamine (10^?7–10^?4 M) induced rapid increases in [Ca²⁺]_i in suspended cells as monitored with the fluorescent calcium indicator fura-2. In [³H]myoinositol-labeled cells, both thrombin (3 U/ml)- and histamine (10^?4 M)-induced IP₃ increases (195% ± 6% and 98% ± 4%, respectively) occurred in less than 15 sec and were temporally correlated with [Ca²⁺]_i increases. Brief incubations (5–60 min) with different protein kinase C activators [4-β-phorbol 12-myristate 13-acetate (1–100 nM), mezerein (100 nM), and sn-1,2 dioctanoylglycerol (0.1–10 μM)] attenuated agonist-induced increases in [Ca²⁺]_i. These compounds also inhibited thrombin- and histaminestimulated IP₃ formation, thus suggesting a tight coupling between phospholipase C activation and calcium flux in cultured HEC. Overall, these observations suggest that the pathway linking receptors to phospholipase C stimulation in human endothelial cells is sensitive to protein kinase C activation.     

Prostaglandin-stimulated second messenger signaling in bone-derived endothelial cells is dependent on confluency in culture

New bone formation is associated with an increase in blood flow by the invasion of capillaries. Endothelial cells that line the capillaries can produce paracrine factors that affect bone growth and development, and in turn, could be affected by products produced by bone cells, in particular the osteoblasts. Since osteoblasts produce prostaglandins E₂ and F_2α (PGE₂, PGF_2α), it was investigated if these PGs were agonists to bone-derived endothelial cells (BBE) by assessing changes in cAMP and free cytosolic calcium concentration ([Ca²⁺]i) second messenger generation. We found that confluent cultures of BBE cells, a clonal endothelial cell line derived from bovine sternal bone, responded to 1 μM PGE₂ by an increase in cAMP. PGF_2α at the same concentration was less potent in stimulating an increase in cAMP production in confluent BBE cells. Subconfluent cells with a morphology similar to that of fibroblastic cells were not as sensitive to PGE₂-stimulated cAMP generation. PGF_2α failed to elicit any cAMP production in subconfluent cultures. PGE₂ and PGF_2α both stimulated an increase in [Ca²⁺]i concentration in a dose-dependent manner. The potency of PGE₂ was similar to that of PGF_2α in stimulating an increase in [Ca²⁺]i. The Ca²⁺ response was mostly independent of extracellular Ca⁺, was unchanged even with prior indomethacin treatment, was unaffected by caffeine pretreatment, but was abolished subsequent to thapsigargin pretreatment. The PG-induced increase in [Ca²⁺]i was also dependent on the confluency of the cells. In a subconfluent state, the responses to PGE₂ or PGF_2α were either negligible, or only small increases in [Ca²⁺]i were noted with high concentrations of these two PGs. Consistent, dose-dependent increases in [Ca²⁺]i were stimulated by these PGs only when the cells were confluent and had a cobblestoned appearance. Since it was previously demonstrated that BBE cells respond to parathyroid hormone (PTH) by the production of cAMP, we tested if bovine PTH(1-34) amide bPTH(1—34) also increased [Ca²⁺]i in these cells. No change in [Ca²⁺]i was found in response to bPTH (1—34), although bPTH (1—34) stimulated a nine to tenfold increase in cAMP. We conclude that BBE cells respond to PGE₂ and PGF_2α but not to bPTH(1—34) by an increase in [Ca²⁺]i probably secondary to stimulation of phospholipase C and that the cAMP and [Ca²⁺]i second messenger responses in BBE cells are dependent on the state of confluency of the cells. © 1994 Wiley-Liss, Inc.     

Infrared neural stimulation induces intracellular Ca2+ release mediated by phospholipase C

The influence of infrared laser pulses on intracellular Ca²⁺ signaling was investigated in neural cell lines with fluorescent live cell imaging. The probe Fluo‐4 was used to measure Ca²⁺ in HT22 mouse hippocampal neurons and nonelectrically excitable U87 human glioblastoma cells exposed to 50 to 500 ms infrared pulses at 1470 nm. Fluorescence recordings of Fluo‐4 demonstrated that infrared stimulation induced an instantaneous intracellular Ca²⁺ transient with similar dose‐response characteristics in hippocampal neurons and glioblastoma cells (half‐maximal effective energy density EC₅₀ of around 58 J.cm^?2). For both type of cells, the source of the infrared‐induced Ca²⁺ transients was found to originate from intracellular stores and to be mediated by phospholipase C and IP₃‐induced Ca²⁺ release from the endoplasmic reticulum. The activation of phosphoinositide signaling by IR light is a new mechanism of interaction relevant to infrared neural stimulation that will also be widely applicable to nonexcitable cell types. The prospect of infrared optostimulation of the PLC/IP₃ cell signaling cascade has many potential applications including the development of optoceutical therapeutics.      

Continuous measurement of changes in intracellular calcium concentration in mouse splenic T cells attached to a glass substrate

Mitogen- and isoproterenol-induced changes of [Ca²⁺]_i in T cells attached to a glass substrate were examined. Murine (C57BL/6) splenic T cells were attached to coverslips or 35-mm dishes (MatTek) precoated with Cell Tak® (3.5 µg/cm²). The cells were then loaded with fluorescent dye (2 µg/ml of fura2-AM or fluo3-AM) and changes in [Ca²⁺]_i in a population of cells (using a spectrofluorometer) or in single cells (using a confocal microscope) were measured during continuous superfusion. Population measurements of [Ca²⁺]_i demonstrated that concanavalin A (Con A, 2 or 5 µg/ml) caused an increase in [Ca²⁺]_i that rose to a peak and then declined to a steady state. The concentration-response relationship (0.05–5 µg/ml) had an EC₅₀ of 0.3 µg/ml. Isoproterenol decreased the Con A-induced elevation of steady state [Ca²⁺]_i. In single cell studies, the increase in [Ca²⁺]_i in response to Con A typically occurred in about 50% of the cells in a microscope field, and the delay before activation varied among cells. Taken together, these data demonstrate that Cell Tak® can be used to attach T cells to glass coverslips and will be useful for the study of signaling mechanisms in T cells.     

Differential Responses of Cultured MC3T3-E1 Cells to Dynamic and Static Stimulated Effect of Microgravity in Cell Morphology,Cytoskeleton Structure and Ca²⁺ Signaling

Random positioning machine (RPM) and diamagnetic levitation are two essential ground-based methods used to stimulate the effect of microgravity in space life science research. However, the force fields generated by these two methods are fundamentally different, as RPM generates a dynamic force field acting on the surface in contact with supporting substrate, whereas diamagnetic levitation generates a static force field acting on the whole body volume of the object (e.g. cell). Surprisingly, it is hardly studied whether these two fundamentally different force fields would cause different responses in mammalian cells. Thus we exposed cultured MC3T3-E1 osteoblasts to either dynamically stimulated effect of microgravity (d-µg) with RPM or statically stimulated effect of microgravity (s-µg) with diamagnetic levitation, respectively, for 3 h. Subsequently, the cells were examined for changes in cell morphology, cytoskeleton (CSK) structure and Ca²⁺ signaling. The results show that compared to the condition of normal gravity (1g), both d-µg and s-µg resulted in decrease of cell area and disruption of the microfilaments and microtubules in MC3T3-E1 cells, but cells under d-µg were more smooth and round while those under s-µg exhibited more protrusions. The decrease of cell area and disruption of microfilaments and microtubules induced by d-µg but not s-µg were rescued by inhibition of the stretch-activated channel by gadolinium chloride (Gd). Inhibition of calmodulin (CaM) by inhibitor, W-7, promoted the effects of s-µg on cell area and CSK filaments, but inhibition of calmodulin-dependent protein kinase (CaMK) by inhibitor, KN-93, weakened d-µg-induced effects on cell area and cytoskeleton. In addition, both d-µg and s-µg decreased the CaM expression and CaMKⅡ activity in MC3T3-E1 cells. Furthermore, s-µg resulted in decrease of the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in MC3T3-E1 cells, which was reversed by disrupting microfilaments with cytochalasin B (CytB). Instead, d-µg induced increase of [Ca²⁺]_i, which was inhibited by Gd. Taken together these data suggest that dynamic and static stimulated microgravity cause different responses in MC3T3-E1 cells. The dynamic force field acts on stretch-activated channels to induce microfilaments disruption and Ca²⁺ influx in MC3T3-E1 cells whereas the static force field directly induces microfilament disruption, which in turn decreases the [Ca²⁺]_i in MC3T3-E1 cells. Such findings may have important implications to better understanding microgravity related cellular events and their applications.     

Human platelets use a cytosolic Ca2+ nanodomain to activate Ca2+-dependent shape change independently of platelet aggregation

Human platelets use a rise in cytosolic Ca²⁺ concentration to activate all stages of thrombus formation, however, how they are able to decode cytosolic Ca²⁺ signals to trigger each of these independently is unknown. Other cells create local Ca²⁺ signals to activate Ca²⁺-sensitive effectors specifically localised to these subcellular regions. However, no previous study has demonstrated that agonist-stimulated human platelets can generate a local cytosolic Ca²⁺ signal. Platelets possess a structure called the membrane complex (MC) where the main intracellular calcium store, the dense tubular system (DTS), is coupled tightly to an invaginated portion of the plasma membrane called the open canalicular system (OCS). Here we hypothesised that human platelets use a Ca²⁺ nanodomain created within the MC to control the earliest phases of platelet activation. Dimethyl-BAPTA-loaded human platelets were stimulated with thrombin in the absence of extracellular Ca²⁺ to isolate a cytosolic Ca²⁺ nanodomain created by Ca²⁺ release from the DTS. In the absence of any detectable rise in global cytosolic Ca²⁺ concentration, thrombin stimulation triggered Na⁺/Ca²⁺ exchanger (NCX)-dependent Ca²⁺ removal into the extracellular space, as well as Ca²⁺-dependent shape change in the absence of platelet aggregation. The NCX-mediated Ca²⁺ removal was dependent on the normal localisation of the DTS, and immunofluorescent staining of NCX3 demonstrated its localisation to the OCS, consistent with this Ca²⁺ nanodomain being formed within the MC. These results demonstrated that human platelets possess a functional Ca²⁺ nanodomain contained within the MC that can control shape change independently of platelet aggregation.     

Intracellular Ca2+ Inhibits Smooth Muscle L-Type Ca2+ Channels by Activation of Protein Phosphatase Type 2B and by Direct Interaction with the Channel

Modulation of L-type Ca²⁺ channels by tonic elevation of cytoplasmic Ca²⁺ was investigated in intact cells and inside-out patches from human umbilical vein smooth muscle. Ba²⁺ was used as charge carrier, and run down of Ca²⁺ channel activity in inside-out patches was prevented with calpastatin plus ATP. Increasing cytoplasmic Ca²⁺ in intact cells by elevation of extracellular Ca²⁺ in the presence of the ionophore A23187 inhibited the activity of L-type Ca²⁺ channels in cell-attached patches. Measurement of the actual level of intracellular free Ca²⁺ with fura-2 revealed a 50% inhibitory concentration (IC₅₀) of 260 nM and a Hill coefficient close to 4 for Ca²⁺- dependent inhibition. Ca²⁺-induced inhibition of Ca²⁺ channel activity in intact cells was due to a reduction of channel open probability and availability. Ca²⁺-induced inhibition was not affected by the protein kinase inhibitor H-7 (10 μM) or the cytoskeleton disruptive agent cytochalasin B (20 μM), but prevented by cyclosporin A (1 μg/ ml), an inhibitor of protein phosphatase 2B (calcineurin). Elevation of Ca²⁺ at the cytoplasmic side of inside-out patches inhibited Ca²⁺ channels with an IC₅₀ of 2 μM and a Hill coefficient close to unity. Direct Ca²⁺-dependent inhibition in cell-free patches was due to a reduction of open probability, whereas availability was barely affected. Application of purified protein phosphatase 2B (12 U/ml) to the cytoplasmic side of inside-out patches at a free Ca²⁺ concentration of 1 μM inhibited Ca²⁺ channel open probability and availability. Elevation of cytoplasmic Ca²⁺ in the presence of PP2B, suppressed channel activity in inside-out patches with an IC₅₀ of ∼380 nM and a Hill coefficient of ∼3; i.e., characteristics reminiscent of the Ca²⁺ sensitivity of Ca²⁺ channels in intact cells. Our results suggest that L-type Ca²⁺ channels of smooth muscle are controlled by two Ca²⁺-dependent negative feedback mechanisms. These mechanisms are based on (a) a protein phosphatase 2B-mediated dephosphorylation process, and (b) the interaction of intracellular Ca²⁺ with a single membrane-associated site that may reside on the channel protein itself.     

High extracellular calcium attenuates adipogenesis in 3T3-L1 preadipocytes

We studied the effect of extracellular Ca²⁺ concentration ([Ca²⁺]_e) on adipocyte differentiation. Preadipocytes exposed to continuous [Ca²⁺]_e higher than 2.5 mmol/l accumulated little or no cytoplasmic lipid compared to controls in 1.8 mmol/l [Ca²⁺]_e. Differentiation was monitored by Oil Red O staining of cytoplasmic lipid and triglyceride assay of accumulated lipid, by RT-PCR analysis of adipogenic markers, and by the activity of glycerol-3-phosphate dehydrogenase (GPDH). Elevated [Ca²⁺]_e inhibited expression of peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein α, and steroid regulatory binding element protein. High [Ca²⁺]_e significantly inhibited differentiation marker expression including adipocyte fatty acid binding protein, and GPDH. The decrease in Pref-1 expression that accompanied differentiation also was prevented by high [Ca²⁺]_e. Treatment of 3T3-L1 cells with high [Ca²⁺]_e did not significantly affect cell number or viability and did not trigger apoptosis. Levels of intracellular Ca⁺² remained unchanged in various [Ca²⁺]_e. Treatment of 3T3-L1 with pertussis toxin (PTX) partially restored lipid accumulation and increased differentiation markers in cells treated with 5 mmol/l [Ca²⁺]_e. ‘Classical’ parathyroid cell Ca²⁺ sensing receptors (CaSR) were not detected either by RT-PCR or by Western blotting. These results suggest that continuos exposure to high [Ca²⁺]_e inhibits preadipocyte differentiation and that this may involve a G-protein-coupled mechanism mediated by a novel Ca²⁺ sensor or receptor.     

Stimulation of prostaglandin E2 (PGE2) production by arachidonic acid, oestrogen and parathyroid hormone in MG-63 and MC3T3-E1 osteoblast-like cells

Polyunsaturated fatty acids (PUFAs) as well as oestrogen (E2) and parathyroid hormone (PTH) affect bone cells. The aim of the study was to determine whether arachidonic acid (AA), E2, and PTH increase prostaglandin E₂ (PGE₂) synthesis in MG-63 and MC3T3-E1 osteoblastic cells and the level of mediation by COX-1 and COX-2. PGE₂ levels were determined in the conditioned culture media of MG-63 and MC3T3-E1 osteoblasts after exposure to AA, PTH and E2. Cells were pre-incubated in some experiments with the unselective COX inhibitor indomethacin or the COX-2 specific blocker NS-398. Indirect immunofluorescence was performed on MG-63 cells to detect the presence and location of the two enzymes involved. AA increased PGE₂ secretion in both cell lines; production by MC3T3-E1 cells, however, was significantly higher than that of MG-63 cells. This could be due to autoamplification via the EP₁ subtype of PGE receptors in mouse MC3T3-E1 osteoblasts. Both COX-1 and COX-2 affected the regulation of PGE₂ synthesis in MG-63 cells. E2 had no effect on PGE₂ secretion in both cell lines, while PTH caused a slight increase in PGE₂ synthesis in the MG-63 cell line.     

Mechanisms of STIM1 Activation of Store-Independent Leukotriene C4-Regulated Ca2+ Channels

We recently showed, in primary vascular smooth muscle cells (VSMCs), that the platelet-derived growth factor activates canonical store-operated Ca²⁺ entry and Ca²⁺ release-activated Ca²⁺ currents encoded by Orai1 and STIM1 genes. However, thrombin activates store-independent Ca²⁺ selective channels contributed by both Orai3 and Orai1. These store-independent Orai3/Orai1 channels are gated by cytosolic leukotriene C₄ (LTC₄) and require STIM1 downstream LTC₄ action. However, the source of LTC₄ and the signaling mechanisms of STIM1 in the activation of this LTC₄-regulated Ca²⁺ (LRC) channel are unknown. Here, we show that upon thrombin stimulation, LTC₄ is produced through the sequential activities of phospholipase C, diacylglycerol lipase, 5-lipo-oxygenease, and leukotriene C₄ synthase. We show that the endoplasmic reticulum-resident STIM1 is necessary and sufficient for LRC channel activation by thrombin. STIM1 does not form sustained puncta and does not colocalize with Orai1 either under basal conditions or in response to thrombin. However, STIM1 is precoupled to Orai3 and Orai3/Orai1 channels under basal conditions as shown using Forster resonance energy transfer (FRET) imaging. The second coiled-coil domain of STIM1 is required for coupling to either Orai3 or Orai3/Orai1 channels and for LRC channel activation. We conclude that STIM1 employs distinct mechanisms in the activation of store-dependent and store-independent Ca²⁺ entry pathways.     

STIM1 and Orai1 mediate thrombin-induced Ca2+ influx in rat cortical astrocytes

In astrocytes, thrombin leads to cytoplasmic Ca²⁺ elevations modulating a variety of cytoprotective and cytotoxic responses. Astrocytes respond to thrombin stimulation with a biphasic Ca²⁺ increase generated by an interplay between ER-Ca²⁺ release and store-operated Ca²⁺ entry (SOCE). In many cell types, STIM1 and Orai1 have been demonstrated to be central components of SOCE. STIM1 senses the ER-Ca²⁺ depletion and binds Orai1 to activate Ca²⁺ influx. Here we used immunocytochemistry, overexpression and siRNA assays to investigate the role of STIM1 and Orai1 in the thrombin-induced Ca²⁺ response in primary cultures of rat cortical astrocytes. We found that STIM1 and Orai1 are endogenously expressed in cortical astrocytes and distribute accordingly with other mammalian cells. Importantly, native and overexpressed STIM1 reorganized in puncta under thrombin stimulation and this reorganization was reversible. In addition, the overexpression of STIM1 and Orai1 increased by twofold the Ca²⁺ influx evoked by thrombin, while knockdown of endogenous STIM1 and Orai1 significantly decreased this Ca²⁺ influx. These results indicate that STIM1 and Orai1 underlie an important fraction of the Ca²⁺ response that astrocytes exhibit in the presence of thrombin. Thrombin stimulation in astrocytes leads to ER-Ca²⁺ release which causes STIM1 reorganization allowing the activation of Orai1 and the subsequent Ca²⁺ influx.     

Potential mechanisms of cytosolic calcium modulation in interferon-γ treated U937 cells

Interferon-γ (IFN-γ) at a concentration of 50 U/ml increased internal Ca²⁺ in the monocyte-like cell line U937 by about 100% within 3 min of addition, as determined by indo-1 fluorescence. This IFN-γ-induced increase was reduced to 30–40% of basal (Ca²⁺)_i by the addition of diltiazem (1 uM) or incubation in Ca²⁺-free buffer. A crude membrane preparation obtained by differential centrifugation of sonicated U937 cells possessed Ca²⁺-ATPase activity (10 nmol ATP hydrolyzed/min/mg protein at 30 C) and sequestered Ca²⁺ to a level of 8 nmol/mg protein in 30 min. Addition of inositol trisphophate (IP₃) (10 uM) after accumulation of Ca²⁺ resulted in release of a portion of the sequestered Ca²⁺ within 30 s, which was then resequestered. Although mitochondrial contamination was indicated by partial inhibition of Ca²⁺ uptake by oligomycin A, this mitochondrial inhibitor had no effect on the IP₃-induced Ca²⁺ release. These results suggest that the increase in U937 cell cytoplasmic Ca²⁺ induced by IFN-γ results from both intacellular redistribution of Ca²⁺, probably via polyphosphoinositide metabolism, and the entry of extracellular Ca²⁺ through slow channels.     

Arachidonic acid and its metabolites increase cytosolic free calcium in bovine luteal cells

We studied the effects of arachidonic acid and its metabolites on intracellular free calcium concentrations ([Ca²⁺]i) in highly purified bovine luteal cell preparations. Corpora lutea were collected from Holstein heifers between days 10 and 12 of the estrous cycle. The cells were dispersed and small and large cells were separated by unit gravity sedimentation and flow cytometry. The [Ca²⁺]i was determined by spectrofluorometry in luteal cells loaded with the fluorescent Ca²⁺ probe. Fura-2. Arachidonic acid elicited a dose-dependent increase in [Ca²⁺]i in both small and large luteal cells, having an effect at concentrations as low as 5μM; and was maximally effective at 50μM. Several other fatty acids failed to exert a similar response. Addition of nordihydroguaiaretic acid (NDGA) or indomethacin failed to suppress the effects of arachidonic acid. In fact, the presence of both inhibitors resulted in increases of [Ca²⁺]i, with NDGA exerting a greater stimulation of [Ca²⁺i than indomethacin. Prostaglandin F_2α (PGF_2α) as well as prostaglandin E₂ (PGE₂) increased [Ca²⁺ in the small luteal cells. These results support the idea that arachidonic acid exerts a direct action in mobilizing [Ca²⁺]i, in the luteal cells. Furthermore, they demonstrate that the cyclooxygenase (PGF_2α and PGE₂) and lipoxygenase products of arachidonic acid metabolism also play a role in increasing [Ca²⁺]i in bovine luteal cells. Since the bovine corpus luteum contains large quantities of arachidonic acid, these findings suggest that this compound may regulate calcium-dependent functions of the corpus luteum, including steroid and peptide hormone production and secretion.     

Effects of bradykinin on Ca2+ mobilization and prostaglandin E2 release in human periodontal ligament cells.

In fura-2-loaded human periodontal ligament (HPDL) cells, bradykinin induced a rapidly transient increase and subsequently sustained increase in cytosolic Ca²⁺ ([Ca²⁺]_i). When external Ca²⁺ was chelated by EGTA, the transient peak of [Ca²⁺]_i was reduced and the sustained level was abolished, implying the Ca²⁺ mobilization consists of intracellular Ca²⁺ release and Ca²⁺ influx. Thapsigargin, a specific Ca²⁺-ATPase inhibitor for inositol 1,4,5-trisphosphate (1,4,5-1P₃)-sensitive Ca²⁺ pool, induced an increase in [Ca²⁺]_i in the absence of external Ca²⁺. After depletion of the intracellular Ca²⁺ pool by thapsigargin, the increase in [Ca²⁺]_i induced by bradykinin was obviously reduced. Bradykinin also stimulated formation of inositol polyphosphates including 1,4,5-IP₃. These results suggest that bradykinin stimulates intracellular Ca²⁺ release from the 1,4,5-1P₃-sensitive Ca²⁺ pool. Bradykinin stimulated prostaglandin E₂ (PGE₂) release in the presence of external Ca²⁺, but not in the absence of external Ca²⁺. Ca²⁺ ionophore A23187 and thapsigargin evoked the release of PGE₂ in the presence of external Ca²⁺ despite no activation of bradykinin receptors. These results indicate that bradykinin induces Ca²⁺ mobilization via activation of phospholipase C and PGE₂ release caused by the Ca²⁺ influx in HPDL cells.