The role of divalent cations in the regulation of microtubule assembly. In vivo studies on microtubules of the heliozoan axopodium using the ionophore A23187

Low concentrations of calcium and magnesium ions have been shown to influence microtubule assembly in vitro. To test whether these cations also have an effect on microtubules in vivo, specimens of Actinosphaerium eichhorni were exposed to different concentrations of Ca++ and Mg++ and the divalent cation ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Experimental degradation and reformation of axopodia were studied by light and electron microscopy. In the presence of Ca++ and the ionophore axopodia gradually shorten, the rate of shortening depending on the concentrations of Ca++ and the ionophore used. Retraction of axopodia was observed with a concentration of Ca++ as low as 0.01 mM. After transfer to a Ca++-free solution containing EGTA, axopodia re-extend; the initial length is reached after about 2 h. Likewise, reformation of axopodia of cold-treated organisms is observed only in solutions of EGTA or Mg++, whereas it is completely inhibited in a Ca++ solution. Electron microscope studies demonstrate degradation of the axonemal microtubular array in organisms treated with Ca++ and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. No alteration was observed in organisms treated with Mg++ or EGTA plus ionophore. The results suggest that, in the presence of the ionophore, formation of axonemal microtubules can be regulated by varying the Ca++ concentration in the medium. Since {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 tends to equilibrate the concentrations of divalent cations between external medium and cell interior, it is likely that microtubule formation invivo is influenced by micromolar concentrations of Ca++. These concentrations are low enough to be of physiological significance for a role in the regulation of microtubule assembly in vivo.     

Epidermal growth factor-induced centrosomal separation: mechanism and relationship to mitogenesis

Using a rabbit antibody to MAP1 to stain centrosomes we have studied the mechanism by which epidermal growth factor (EGF) induces centrosomal separation in HeLa cells. The response is rapid, being detectable within 20 min after EGF (100 ng/ml) addition and by 4 h 40% of logarithmically growing cells and greater than 70% of cells synchronized at G1/S with 1 mM hydroxyurea show centrosomes separated by more than one diameter. A concentration of 0.05 ng/ml of EGF induces significant separation in synchronized cells (5-9% control vs. 20% with EGF at 0.05 ng/ml) and 0.1 to 0.5 ng/ml induces a half maximal response. Centrosomal separation is blocked by energy inhibitors, trifluoperazine, chlorpromazine, and W-7, cytochalasins B and D, and taxol, and is stimulated or enhanced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, colchicine, and oncodazole. Trifluoperazine, W-7, cytochalasin D, and taxol also block DNA synthesis in response to EGF as measured by autoradiography using [3H]thymidine. Our hypothesis based upon these results is that EGF, by raising the free calcium level, activates calmodulin, which stimulates contraction of microfilaments attached to the centrosome, pulling the daughter centrosome apart. EGF may also induce depolymerization or detachment of microtubules in the vicinity of the centrosome which ordinarily serve to maintain its position and inhibit separation. Centrosomal separation may be a key event in triggering DNA synthesis in response to EGF and colchicine.     

Calcium in epithelial cell contraction

Epithelial morphogenesis in many organs involves asymmetric microfilament-mediated cellular contractions. Similar contractions, in terms of ultrastructure and cytochalasin B sensitivity, can be induced in the carcinoma cell line C-4II in culture. This line was used to compare total intracellular calcium levels ([Ca]i) in contracted monolayer fragments and in control cultures, and to determine whether epithelial cell contraction depends on influx of extracellular Ca. [Ca]i, defined as Ca not displaceable by lanthanum, was measured by atomic absorption spectrophotometry. Degrees of contraction were determined from shape changes of monolayer fragments. Detachment from the growth surface initiated cellular contractions and caused an immediate increase in [Ca]i, from 1.0 to 4.0-5.0 micrograms Ca/mg protein in early confluent cultures, and from 0.3 to 1.0-2.0 micrograms Ca/mg protein in crowded cultures. This increase was followed by a gradual decline in [Ca]i, though Ca levels remained higher than in controls and contraction progressed for 30 min. Contraction was inhibited completely by cold (7 degrees C) and by Ca-free medium, and in a dose-dependent manner by papaverine (2.5 x 10(-6) M-2.5 x 10(-4) M), lanthanum (1.0 x 10(-6) M-1.0 x 10(-4) M); and D-600 (1.0-2.0 x 10(- 4) M). The Ca ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 had no effect at 5.0 x 10(-6) M and was inhibitory at higher concentrations. The results provided direct evidence for increased [Ca]i in contracting epithelial cells, and suggest that Ca influx is required for such contraction to take place.     

Calcium control of ciliary reversal in ionophore-treated and ATP- reactivated comb plates of ctenophores

Previous work showed that ctenophore larvae swim backwards in high-KCl seawater, due to a 180 degrees reversal in the direction of effective stroke of their ciliary comb plates (Tamm, S. L., and S. Tamm, 1981, J. Cell Biol., 89: 495-509). Ion substitution and blocking experiments indicated that this response is Ca2+ dependent, but comb plate cells are innervated and presumably under nervous control. To determine whether Ca2+ is directly involved in activating the ciliary reversal mechanism and/or is required for synaptic triggering of the response, we (a) determined the effects of ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and Ca2+ on the beat direction of isolated nerve-free comb plates dissociated from larvae by hypotonic, divalent cation-free medium, and (b) used permeabilized ATP- reactivated models of comb plates to test motile responses to known concentrations of free Ca2+. We found that 5 microM {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and 10 mM Ca2+ induced dissociated comb plate cells to beat in the reverse direction and to swim counterclockwise in circular paths instead of in the normal clockwise direction. Detergent/glycerol-extracted comb plates beat actively in the presence of ATP, and reactivation was reversibly inhibited by micromolar concentrations of vanadate. Free Ca2+ concentrations greater than 10(-6)M caused reversal in direction of the effective stroke but no significant increase in beat frequency. These results show that ciliary reversal in ctenophores, like that in protozoa, is activated by an increase in intracellular free Ca2+ ions. This allows the unique experimental advantages of ctenophore comb plate cilia to be used for future studies on the site and mechanism of action of Ca2+ in the regulation of ciliary motion.     

Diffusion rates of cell surface antigens of mouse-human heterokaryons. III. Regulation of lateral diffusion rates by calcium ions

In mouse-human heterokaryons, the lateral diffusion of major histocompatibility (MHC) antigens in the plasma membrane is enhanced by treatment of parent cells with ouabain. Ouabain treatment is ineffective if the medium lacks calcium ion, or if Verapamil, a blocker of calcium channels, is present. The divalent ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 also enhances lateral diffusion of MHC antigens, to the same extent as ouabain, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 is effective only if calcium is present in the medium. Thus it appears that increased levels of cell calcium release constraints to lateral diffusion of MHC antigens.     

Stimulation of catecholamine secretion from cultured chromaffin cells by an ionophore-mediated rise in intracellular sodium

The significance of intracellular Na+ concentration in catecholamine secretion of cultured bovine adrenal chromaffin cells was investigated using the monovalent carboxylic ionophore monensin. This ionophore, which is known to mediate a one-for-one exchange of intracellular K+ for extracellular Na+, induces a slow, prolonged release of catecholamines which, at 6 h, amounts of 75-90% of the total catecholamines; carbachol induces a rapid pulse of catecholamine secretion of 25-35%. Although secretory granule numbers appear to be qualitatively reduced after carbachol, multiple carbachol, or Ba2+ stimulation, overall granule distribution remains similar to that in untreated cells. Monensin-stimulated catecholamine release requires extracellular Na+ but not Ca2+ whereas carbachol-stimulated catecholamine release requires extracellular Ca2+ and is partially dependent on extracellular Na+. Despite its high selectivity for monovalent ions, monensin is considerably more effective in promoting catecholamine secretion than the divalent ionophores, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and ionomycin, which mediate a more direct entry of extracellular Ca2+ into the cell. We propose that the monensin-stimulated increase in intracellular Na+ levels causes an increase in the availability of intracellular Ca2+ which, in turn, stimulates exocytosis. This hypothesis is supported by the comparable stimulation of catecholamine release by ouabain which inhibits the outwardly directed Na+ pump and thus permits intracellular Na+ to accumulate. The relative magnitudes of the secretion elicited by monensin, carbachol, and the calcium ionophores, are most consistent with the hypothesis that, under normal physiological conditions, Na+ acts by decreasing the propensity of Ca2+- sequestering sites to bind the Ca2+ that enters the cell as a result of acetylcholine stimulation.     

Gap junction dynamics: reversible effects of divalent cations

Reversible changes in gap junction structure similar to those previously seen to parallel electrical uncoupling (9, 33, 34) are produced by treating with Ca++ or Mg++ gap junctions isolated in EDTA from calf lens fibers. The changes, characterized primarily by a switch from disordered to crystalline particle packings, occur at a [Ca++] of 5 x 10(-7) M or higher and a [Mg++] of 1 x 10(-3) M or higher and can be reversed by exposing the junctions to Ca++- and Mg++-free EGTA solutions. Similar changes are obtained in junctions of rat stomach epithelia incubated at 37 degrees C in well-oxygenated Tyrode''s solutions containing a Ca++ ionophore ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187). Deep etching experiments on isolated lens junctions show that the true cytoplasmic surface of the junctions (PS face) is mostly bare, suggesting that the particles may not be connected to cytoskeletal elements. A hypothesis is proposed suggesting a mechanism of particle aggregation and channel narrowing based on neutralization of negative charges by divalent cations or H+.     

Epidermal growth factor receptor-mediated cell motility: phospholipase C activity is required,but mitogen-activated protein kinase activity is not sufficient for induced cell movement

We recently have demonstrated that EGF receptor (EGFR)-induced cell motility requires receptor kinase activity and autophosphorylation (P. Chen, K. Gupta, and A. Wells. 1994. J. Cell Biol. 124:547-555). This suggests that the immediate downstream effector molecule contains a src homology-2 domain. Phospholipase C gamma (PLC gamma) is among the candidate transducers of this signal because of its potential roles in modulating cytoskeletal dynamics. We utilized signaling-restricted EGFR mutants expressed in receptor devoid NR6 cells to determine if PLC activation is necessary for EGFR-mediated cell movement. Exposure to EGF (25 nM) augmented PLC activity in all five EGFR mutant cell lines which also responded by increased cell movement. Basal phosphoinositide turnover was not affected by EGF in the lines which do not present the enhanced motility response. The correlation between EGFR-mediated cell motility and PLC activity suggested, but did not prove, a causal link. A specific inhibitor of PLC, {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (1 microM) diminished both the EGF- induced motility and PLC responses, while its inactive analogue {"type":"entrez-nucleotide","attrs":{"text":"U73343","term_id":"1688125","term_text":"U73343"}}U73343 had no effect on these responses. Both the PLC and motility responses were decreased by expression of a dominant-negative PLC gamma-1 fragment in EGF-responsive infectant lines. Lastly, anti-sense oligonucleotides (20 microM) to PLC gamma-1 reduced both responses in NR6 cells expressing wild-type EGFR. These findings strongly support PLC gamma as the immediate post receptor effector in this motogenic pathway. We have demonstrated previously that EGFR-mediated cell motility and mitogenic signaling pathways are separable. The point of divergence is undefined. All kinase-active EGFR mutants induced the mitogenic response while only those which are autophosphorylated induced PLC activity. {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 did not affect EGF-induced thymidine incorporation in these motility-responsive infectant cell lines. In addition, the dominant-negative PLC gamma-1 fragment did not diminish EGF-induced thymidine incorporation. All kinase active EGFR stimulated mitogen-activated protein (MAP) kinase activity, regardless of whether the receptors induced cell movement; this EGF-induced MAP kinase activity was not affected by {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 at concentrations that depressed the motility response. Thus, the signaling pathways which lead to motility and cell proliferation diverge at the immediate post-receptor stage, and we suggest that this is accomplished by differential activation of effector molecules.     

Localization of calcium in presynaptic nerve terminals. An ultrastructural and electron microprobe analysis

Ultrastructural techniques and electron probe microanalysis were used to determine whether or not the smooth endoplasmic reticulum (SER) within presynaptic nerve terminals is a Ca-sequestering site. The three- dimensional structure of the SER was determined from serial sections of synaptosomes. The SER consists of flattened cisterns that may branch and are frequently juxtaposed to mitochondria. To investigate intraterminal Ca sequestration, synaptosomes were treated with saponin to disrupt the plasmalemmal permeability barrier. When these synaptosomes were incubated in solutions containing Ca, ATP, and oxalate, electrondense Ca oxalate deposits were found in intraterminal mitochondria, SER cisterns, and large vesicular profiles. Saponin- treated synaptosomes that were incubated in the presence of mitochondrial poisons contained electron-dense deposits within SER cisterns and large vesicular profiles, but very rarely in mitochondria. Similar deposits were observed within saponin-treated synaptosomes that were not post-fixed with OSO4, and within saponin-treated synaptosomes that were prepared for analysis by freeze-substitution. Electron-probe microanalyses of these deposits confirmed the presence of large concentrations of Ca. When oxalate was omitted from the incubation solutions, no electron-dense deposits were present in saponin-treated synaptosomes. In other control experiments, either the Ca ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 or the Ca chelator EGTA was added to the incubation media; electron-dense deposits were very rarely observed within the intraterminal organelles of these saponin-treated synaptosomes. The data indicate that presynaptic nerve terminal SER is indeed a Ca- sequestering organelle.     

Epidermal Growth Factor Stimulates Nuclear Factor-κB Activation and Heme Oxygenase-1 Expression via c-Src,NADPH Oxidase,PI3K,and Akt in Human Colon Cancer Cells

Previous report showed that epidermal growth factor (EGF) promotes tumor progression. Several studies demonstrated that growth factors can induce heme oxygenase (HO)-1 expression, protect against cellular injury and cancer cell proliferation. In this study, we investigated the involvement of the c-Src, NADPH oxidase, reactive oxygen species (ROS), PI3K/Akt, and NF-κB signaling pathways in EGF-induced HO-1 expression in human HT-29 colon cancer cells. Treatment of HT-29 cells with EGF caused HO-1 to be expressed in concentration- and time-dependent manners. Treatment of HT-29 cells with AG1478 (an EGF receptor (EGFR) inhibitor), small interfering RNA of EGFR (EGFR siRNA), a dominant negative mutant of c-Src (c-Src DN), DPI (an NADPH oxidase inhibitor), glutathione (an ROS inhibitor), {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a PI3K inhibitor), and an Akt DN inhibited EGF-induced HO-1 expression. Stimulation of cells with EGF caused an increase in c-Src phosphorylation at Tyr406 in a time-dependent manner. Treatment of HT-29 cells with EGF induced an increase in p47^phox translocation from the cytosol to membranes. The EGF-induced ROS production was inhibited by DPI. Stimulation of cells with EGF resulted in an increase in Akt phosphorylation at Ser473, which was inhibited by c-Src DN, DPI, and LY 294002. Moreover, treatment of HT-29 cells with a dominant negative mutant of IκB (IκBαM) inhibited EGF-induced HO-1 expression. Stimulation of cells with EGF induced p65 translocation from the cytosol to nuclei. Treatment of HT-29 cells with EGF induced an increase in κB-luciferase activity, which was inhibited by a c-Src DN, LY 294002, and an Akt DN. Furthermore, EGF-induced colon cancer cell proliferation was inhibited by Sn(IV)protoporphyrin-IX (snPP, an HO-1 inhibitor). Taken together, these results suggest that the c-Src, NADPH oxidase, PI3K, and Akt signaling pathways play important roles in EGF-induced NF-κB activation and HO-1 expression in HT-29 cells. Moreover, overexpression of HO-1 mediates EGF-induced colon cancer cell proliferation.     

Two distinct mechanisms for redistribution of lymphocyte surface macromolecules. II. Contrasting effects of local anesthetics and a calcium ionophore

In the previous study, lymphocyte surface molecules were separated into two subsets depending on whether capping was associated was associated with redistribution of cytoplasmic myosin. In the present study, the effects of the local anesthetic chlorpromazine and of the Ca2+ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 were compared. Both drugs affected the surface redistribution of immunoglobulin (Ig), Fc receptors, and the TL antigen- -molecules that appear to cap by association with microfilaments--but had no effect on the Thy.1 (theta) and H2 antigens--molecules that cap slowly, apparently unlinked to microfilament function. The capping of Ig, Fc receptor, and TL was inhibited while that of H2 and theta was not. Both drugs reversed the Ig Fc receptor, and TL caps but not the H2 and theta caps. In the former group, the reversal of caps was accompanied by a parallel reversal of the myosin segregated to the cap area. The appearance of myosin after drug treatment varied: chlorpromazine resulted in a diffuse pattern similar to that of normal lymphocytes, whereas {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 produced an array of aggregates and coarse filaments. The results are compatible with the view that two mechanisms for capping exist in the lymphocyte. The Ca2+ ionophore may affect capping of microfilament-dependent caps by producing a systemic activation of contractile proteins while chlorpromazine may act by disrupting a Ca2+-dependent link between surface complexes and the contractile proteins.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

Serine Hydrolase Inhibitors Block Necrotic Cell Death by Preventing Calcium Overload of the Mitochondria and Permeability Transition Pore Formation

Perturbation of calcium signaling that occurs during cell injury and disease, promotes cell death. In mouse lung fibroblasts {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 triggered mitochondrial permeability transition pore (MPTP) formation, lactate dehydrogenase (LDH) release, and necrotic cell death that were blocked by cyclosporin A (CsA) and EGTA. LDH release temporally correlated with arachidonic acid release but did not involve cytosolic phospholipase A₂α (cPLA₂α) or calcium-independent PLA₂. Surprisingly, release of arachidonic acid and LDH from cPLA₂α-deficient fibroblasts was inhibited by the cPLA₂α inhibitor pyrrophenone, and another serine hydrolase inhibitor KT195, by preventing mitochondrial calcium uptake. Inhibitors of calcium/calmodulin-dependent protein kinase II, a mitochondrial Ca²⁺ uniporter (MCU) regulator, also prevented MPTP formation and arachidonic acid release induced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and H₂O₂. Pyrrophenone blocked MCU-mediated mitochondrial calcium uptake in permeabilized fibroblasts but not in isolated mitochondria. Unlike pyrrophenone, the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol and CsA blocked cell death and arachidonic acid release not by preventing mitochondrial calcium uptake but by inhibiting MPTP formation. In fibroblasts stimulated with thapsigargin, which induces MPTP formation by a direct effect on mitochondria, LDH and arachidonic acid release were blocked by CsA and 1-oleoyl-2-acetyl-sn-glycerol but not by pyrrophenone or EGTA. Therefore serine hydrolase inhibitors prevent necrotic cell death by blocking mitochondrial calcium uptake but not the enzyme releasing fatty acids that occurs by a novel pathway during MPTP formation. This work reveals the potential for development of small molecule cell-permeable serine hydrolase inhibitors that block MCU-mediated mitochondrial calcium overload, MPTP formation, and necrotic cell death.     

Enhancement of calcium uptake and phosphatidylinositol turnover by epidermal growth factor in A-431 cells

Epidermal growth factor (EGF) stimulates the incorporation of 32Pi and [3H]inositol into phosphatidylinositol (5-10-fold) in A-431 cells. EGF also stimulates the incorporation of 32Pi into phosphatidic acid (up to 10-fold). These effects are attributed to an acceleration of the turnover of phosphatidylinositol as a consequence of the binding of EGF to its membrane receptor. The extent of the phosphatidylinositol response to EGF parallels the extent of hormone binding. The phosphatidylinositol response to EGF appears to be dependent on an influx of calcium since (a) external calcium is required for the enhancement of phosphatidylinositol turnover, (2) the accumulation of 45Ca by A-431 cells is stimulated by EGF, (3) blockage of calcium influx with LaCl3 inhibits stimulation of phosphatidylinositol turnover, and (4) calcium influx via ionophore A23187 is sufficient to stimulate phosphatidylinositol turnover. Since the binding, internalization, and degradation of 125I-labeled EGF in A-431 cells are unaffected by the omission of calcium from the medium, external calcium and phosphatidylinositol turnover are not necessary for the internalization and degradation of the EGF-receptor complex.     

Stimulation and inhibition of secretion in Paramecium: role of divalent cations

The effects of Ca2+ and Mg2+ on exocytosis in Paramecium tetraurelia cells were examined with light microscopy, freeze fracture (FEM) and transmission electron microscopy (TEM) of thin-sectioned embedded cells. Picric acid-Ca2+-induced secretion in wild type (wt) cells was captured by "quick" fixation with OsO4, and TEM demonstrated membrane fusion occurring before trichocyst matrix (tmx) expansion. Cells stimulated with picric acid in the presence of high extracellular Mg2+ showed very few sites of membrane fusion and no tmx expansion, suggesting that Ca2+ is required for both membrane fusion and tmx expansion. Further information was obtained by comparing secretory responses of wt cells with a temperature-sensitive secretory mutant, nd 9. These cells when grown at the permissive temperature (18 degrees C) possess normal rosettes at the secretory site and secrete in response to picric acid-Ca2+, but when grown at 27 degrees C they lack rosettes and do not secrete (Beisson, J., M. Lefort-Tran, M. Pouphile, M. Rossignol, and B. Satir, 1976, J. Cell Biol., 69:126-143). Quantitation of picric acid-Ca2+-induced secretion revealed that: (a) the number of tmx secreted by wt and nd 9 cells was independent of their cultural growth phase, (b) wt cells secreted the same number of tmx when grown either at 18 or 27 degrees C, and (c) nd 9 18 degrees C cells secreted the same number of tmx as wt 18 or 27 degrees C cells. Wild type and nd 9 cells had the same frequencies of occupied and unoccupied secretory sites as determined by quantitative analysis of freeze-fracture replicas. After stimulation with divalent cation ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 and Ca2+, wt cells showed a significant reduction in the frequency of occupied sites. FEM and TEM studies revealed that {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187-Ca2+ induced tmx expansion and normal fusion of the plasma and trichocyst membranes in wt and nd 9 18 degrees C cells, but induced tmx expansion without concomitant membrane fusion in nd 9 27 degrees C cells. The lack of membrane fusion in nd 9 27 degrees C cells suggests that the molecules represented by rosette particles are required specifically for membrane fusion.     

Vesicle exocytosis stimulated by alpha-latrotoxin is mediated by latrophilin and requires both external and stored Ca2+.

alpha-Latrotoxin (LTX) stimulates massive neurotransmitter release by two mechanisms: Ca2+-dependent and -independent. Our studies on norepinephrine secretion from nerve terminals now reveal the different molecular basis of these two actions. The Ca2+-dependent LTX-evoked vesicle exocytosis (abolished by botulinum neurotoxins) is 10-fold more sensitive to external Ca2+ than secretion triggered by depolarization or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187; it does not, however, depend on the cation entry into terminals but requires intracellular Ca2+ and is blocked by drugs depleting Ca2+ stores and by inhibitors of phospholipase C (PLC). These data, together with binding studies, prove that latrophilin, which is linked to G proteins and inositol polyphosphate production, is the major functional LTX receptor. The Ca2+-independent LTX-stimulated release is not inhibited by botulinum neurotoxins or drugs interfering with Ca2+ metabolism and occurs via pores in the presynaptic membrane, large enough to allow efflux of neurotransmitters and other small molecules from the cytoplasm. Our results unite previously contradictory data about the toxin''s effects and suggest that LTX-stimulated exocytosis depends upon the co-operative action of external and intracellular Ca2+ involving G proteins and PLC, whereas the Ca2+-independent release is largely non-vesicular.     

Ethyl Pyruvate Inhibits HMGB1 Phosphorylation and Release by Chelating Calcium

Ethyl pyruvate (EP), a simple aliphatic ester of pyruvic acid, has been shown to have antiinflammatory effects and to confer protective effects in various pathological conditions. Recently, a number of studies have reported EP inhibits high mobility group box 1 (HMGB1) secretion and suggest this might contribute to its antiinflammatory effect. Since EP is used in a calcium-containing balanced salt solution (Ringer solution), we wondered if EP directly chelates Ca²⁺ and if it is related to the EP-mediated suppression of HMGB1 release. Calcium imaging assays revealed that EP significantly and dose-dependently suppressed high K⁺-induced transient [Ca²⁺]_i surges in primary cortical neurons and, similarly, fluorometric assays showed that EP directly scavenges Ca²⁺ as the peak of fluorescence emission intensities of Mag-Fura-2 (a low-affinity Ca²⁺ indicator) was shifted in the presence of EP at concentrations of ≥7 mmol/L. Furthermore, EP markedly suppressed the {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253"}}A23187-induced intracellular Ca²⁺ surge in BV2 cells and, under this condition, {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253"}}A23187-induced activations of Ca²⁺-mediated kinases (protein kinase Cα and calcium/calmodulin-dependent protein kinase IV), HMGB1 phosphorylation and subsequent secretion of HMGB1 also were suppressed. ({"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253"}}A23187 is a calcium ionophore and BV2 cells are a microglia cell line.) Moreover, the above-mentioned EP-mediated effects were obtained independent of cell death or survival, which suggests that they are direct effects of EP. Together, these results indicate that EP directly chelates Ca²⁺, and that it is, at least in part, responsible for the suppression of HMGB1 release by EP.     

Modulation of the activity of cytosolic phospholipase A2�� (cPLA2��) by cellular sphingolipids and inhibition of cPLA2�� by sphingomyelin

We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and activity of cytosolic phospholipase A2α (cPLA2α) using two Chinese hamster ovary (CHO)-K1-derived mutants deficient in sphingolipid synthesis: LY-B cells defective in the LCB1 subunit of serine palmitoyltransferase for de novo synthesis of sphingolipid species, and LY-A cells defective in the ceramide transfer protein CERT for SM synthesis. When LY-B and LY-A cells were cultured in Nutridoma medium and the sphingolipid level was reduced, the release of AA stimulated by the Ca²⁺ ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 increased 2-fold and 1.7-fold, respectively, compared with that from control cells. The enhancement in LY-B cells was decreased by adding sphingosine and treatment with the cPLA2α inhibitor. When CHO cells were treated with an acid sphingomyelinase inhibitor to increase the cellular SM level, the release of AA induced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 or PAF was decreased. In vitro studies were then conducted to test whether SM interacts directly with cPLA2α. Phosphatidylcholine vesicles containing SM reduced cPLA2α activity. Furthermore, SM disturbed the binding of cPLA2α to glycerophospholipids. These results suggest that SM at the biomembrane plays important roles in regulating the cPLA2α-dependent release of AA by inhibiting the binding of cPLA2α to glycerophospholipids.