Effects of hypothermic storage on intracellular calcium, reactive oxygen species formation, mitochondrial function, motility, and plasma membrane integrity in striped bass (Morone saxatilis) sperm

Experiments were conducted to determine the effect of hypothermic 24 h storage on striped bass sperm cell plasma membrane integrity, free intracellular Ca²⁺ ([Ca²⁺]_i), mitochondrial membrane potential (ΔΨ_m), and reactive oxygen species (ROS) formation (oxidation of hydroethidine to ethidium) as determined by flow cytometry; motion activation and ATP concentration as determined by Luciferin-Luciferase bioluminescence assay. Semen was stored for 1 or 24 h at 4 °C in an O₂ atmosphere undiluted or diluted (one volume semen with 3 volumes diluent) with T350 (20 mM TRIS base-NaCl, 350 mOsm/mL, pH 8) or with seminal plasma in the presence of various treatments. Viability (% cells excluding propidium iodide) approached 100% after 1 h storage in undiluted or diluted semen. After 1 h of storage the [Ca²⁺]_i marker, Fluo-3, was detected in only 3% of sperm cells in undiluted or diluted semen. In contrast to storage for 1 h, after 24 h the incidence Fluo-3 fluorescence intensity was increased (P < 0.05) in > 50% of the viable cells in undiluted and diluted semen along with increased cell death; the presence of 1 mM ethylene glycol tetraacetic acid (EGTA) blocked CaCl₂-induced Fluo-3 fluorescence and cell death. Activation of sperm motility was 82% after 1 h in T350 and decreased (P < 0.05) to 30% after 24 h. However, motility activation failed in the presence of EGTA at 1 or 24 h. During storage ΔΨ_m was not affected by storage time or treatment. In contrast, sperm ATP was greater (P < 0.05) at 1 h than at 24 h and was greater in sperm stored in diluted than undiluted form. While ROS formation was induced by menadione treatment, there was no evidence of storage-induced ROS formation in the absence of menadione. The increased [Ca²⁺]_i found after 24 h indicates a storage induced defect in the maintenance of cellular calcium homeostasis which may be detrimental to sperm activation.     

Role of mitogen-induced calcium influx in the control of the cell cycle in Balb-c 3T3 fibroblasts

The role of mitogen-activated calcium influx from the extracellular medium in the control of cell proliferation was studied in Balb-c 3T3 fibroblasts. Stimulation of serum-deprived, quiescent cells with 10% foetal calf serum (FCS) induced a long-lasting (up to 70 min elevation of intracellular free calcium concentration ([Ca²⁺]_i). Both the sustained [Ca ²⁺]_i increase and the related inward current, described in a previous paper [Lovisolo D. Munaron L. Baccino FM. Bonelli G. (1992) Potassium and calcium currents activated by foetal calf serum in Balb-c 3T3 fibroblasts. Biochim. Biophys. Acta, 1104, 73–82], could be abolished either by chelation of extracellular calcium with EGTA or by SKF 96365, an imidazole derivative that can block receptor-activated calcium channels. The effect of the abolition of these ionic signals on FCS-induced proliferation was investigated by adding either EGTA or SK&F 96365 to the culture medium during the first hours of stimulation of quiescent cells with 10% FCS. As measured after 24 h, a 22% inhibition of growth was observed when SK&F 96365 was added for the first hour, and stronger inhibitions, up to 56%, were obtained by adding the blocker for the first 2 or 4 h. Similar effects were observed with addition of 3 mM EGTA, though the inhibition was less marked for the 4 h treatment. By contrast, incubation with either substance in the next 4 h of serum stimulation did not influence cell growth, except for a slight inhibition observed when SKF 96365 was applied from the 4^th to the 8^th hour. The reduction in growth resulting from the abolition of the early calcium influx was paralleled by an accumulation of cells in the G₂/M phase. Both growth inhibition and G₂/M accumulation were reversible, since after further 24 h in 10% FCS cells had fully recovered the exponential growth. These data indicate that the early calcium influx seen in response to mitogen stimulation develops on a timescale long enough to play a significant role in cell cycle progression, and that its block in the early G1 phase can lead to a reduction of proliferation by arresting cells in later stages of the cycle.     

On the role of calcium in chemotaxis and oscillations of dictyostelium cells

Migration of differentiated cells to a capillary containing cyclic AMP was enhanced in the presence of 1 mM CaCl₂ and was virtually absent in the presence of 1 mM EGTA. Furthermore, the cells contracted and extended pseudopods to a capillary filled with the calcium ionophore A 23187. At short distances, migration to the tip of the capillary was observed. The ionophore also induced transient decreases of the optical density of suspended cells indicating changes of cell shape. These findings support the hypothesis that cyclic AMP-binding to cell surface receptors causes a local influx of calcium ions. These in turn lead to an increase of the cytosolic calcium concentration and subsequently to an activation of cell migration. Perturbing pulses of the ionophore induced permanent phase shifts of free-running light scattering oscillations. This result indicates that cytosolic calcium is an intrinsic component of the oscillatory system.Based on material presented at the Symposium Intercellular Communication, Stuttgart, September 16–17, 1982     

Role of prostaglandins in calcium-induced corticosteroid secretion by isolated frog interrenal gland

The role of prostaglandins (PGs) in calcium-induced corticosteroid secretion by frog adrenal (interrenal) gland examined using a perifusion technique. Increasing concentrations of CaCl₂ (4–10 mM) stimulated in a dose-dependent manner aldosterone, PGE₂ and 6-keto-PGF_1α production, whereas TXB₂ was not affected. The kinetics of the adrenal response to CaCl₂ indicated that the increase in PG output always preceded that of steroid. Administration of cobalt (4 mM), a calcium-channel inhibitor, blocked the calcium-induced stimulation of PGs and corticosteroids. Infusion of indomethacin (5 × 10⁻⁶M), a specific cyclooxygenase inhibitor, significantly decreased the basal production of PGs and steroids, and prevented the stimulatory effect of CaCl₂ (6 mM). Infusion of the calcium ionophore A 23187 (10⁻⁶ M), for 20 min, induced a marked stimulation of PG and steroid production. Taken together, these data support the notion that biosynthesis of prostaglandins is associated with calcium-induced corticosteroid secretion in frog adrenal cells.     

Cultivation of a desulfurizing bacterium, Mycobacterium strain G3

Various carbon and sulfur sources on the growth and desulfurization activity of Mycobacterium strain G3, which is a dibenzothiophene (DBT)-degrading microorganism, were studied. Ethanol, glucose or glycerol as the sole carbon source and MgSO₄, taurine or dimethyl sulfoxide (DMSO) as the sole sulfur source were suitable for the growth. In addition, desulfurization activity was expressed in medium containing taurine, MgSO₄ or DMSO at 0.1 mM, when 217 mM ethanol was used as the sole carbon source. The highest desulfurization activity was in the stationary phase cells after 5 days' growth, rather than those harvested during active growth, when Mycobacterium G3 was cultivated in medium containing 217 mM ethanol and 0.1 mM MgSO₄. Thus alternative sulfur sources to DBT can be used for the cultivation of this desulfurizing microorganism.     

Effects of taurine and hypotaurine supplementation and ionophore concentrations on post-thaw acrosome reaction of dog spermatozoa

The aim of this work was to study of the effect of the amino acids (AA) taurine (T) and hypotaurine (H) and of different calcium ionophore concentrations on the ability of capacitated frozen-thawed dog sperm to undergo the acrosome reaction (AR). Fifteen ejaculates grouped into five pools were used. Sperm was frozen at a concentration of 80 × 10⁶ sperm cells/mL in the Uppsala Equex extender (UE) supplemented with 25, 50 and 75 mM of either AA. The UE extender without T or H was used as control. After thawing, sperm was capacitated with Canine Capacitation Medium for 20 min. Sperm was then challenged with calcium ionophore A23178 at 0, 2.5 and 10 μM concentration and evaluated for integrity of plasma and acrosome membranes after 5, 15 and 30 min of incubation, utilizing PI/Fitc-PNA fluorescent staining and flow cytometry.Sperm cryopreserved in UE supplemented with 50 mM T (UE 50T) had higher AR rates than sperm cryopreserved with UE 75T, UE 25H and UE 50H, but AR rates were similar to semen frozen with the control extender. Challenges with 2.5 and 10 μM/L of calcium ionophore increased AR in frozen-thawed sperm incubated for 5, 15 and 30 min. The combination of calcium ionophore concentration and incubation time resulting in the highest AR rate was 10 μM and 15 min.     

Influence of calcium on blue-light-induced chloroplast movement in Lemna trisulca L.

The presence of calcium is essential for chloroplast movement induced by blue light in Lemna trisulca L. The regulatory role of calcium was confirmed by the inhibition of chloroplast movement by cytochalasin B and trifluoperazine. The calcium concentration in tissues was modified by ethylene glycol-bis(2-aminoethylether)-N,N,N, N-tetraacetic acid (EGTA), the calcium ionophore A23187 and La³⁺. Only a long period of incubation (12h) in EGTA or La³⁺ caused distrubances in chloroplast movement. This indicates that calcium influx is not essential for chloroplast movement. Those conditions that dramatically changed the internal calcium concentration, either applications of calcium ionophore A23187 and EGTA, or ionophore and La³⁺, markedly decreased the amplitude of response to blue-light pulses. This demonstrates that disturbances of chloroplast movement are observable only when internal stores of calcium are affected by Ca²⁺-antagonists. We suggest that the calcium involved in blue-light-induced chloroplast movement is derived from intracellular stores. The addition of Mg²⁺ to EGTA buffer counteracted its effect, indicating that Mg²⁺, as well as Ca²⁺, might possibly be involved in chloroplast movement.Abbreviations EGTA ethylene glycol-bis(2-aminoethylether)-N,N,N,N-tetraacetic acid - Hepes 4(2-hydroxyethyl-1-piperazine) ethanesulfonic acid - A23187 calcium ionophore We express our gratitude to Professor W. Korohoda for valuable critical comments on this paper and stimulating discussion. We also thank Mr. P. Malec for help in preparing the experiment with trifluoperazine and Mr. A. Waloszek for taking the photographs. We are indebted to Mr. Tim Kline (International House, Krakow, Poland) for improving the English style. This research was supported by grant No. 1042/P2/92/03 from the State Committe for Scientific Research.     

Light regulation of phosphoenolpyruvate carboxylase in barley mesophyll protoplasts is modulated by protein synthesis and calcium, and not necessarily correlated with phosphoenolpyruvate carboxylase kinase activity

The regulation of phosphoenolpyruvate carboxylase (PEPCase, EC. 4.1.1.31) and PEPCase kinase was investigated using barley (Hordeum vulgare L.) mesophyll protoplasts. Incubation of protoplasts in the light resulted in a reduction in the sensitivity of PEPCase to the inhibitor L-malate; PEPCase from protoplasts incubated in the light for 1 h was inhibited 48±2% by 2mM malate, whereas the enzyme from protoplasts incubated for 1 h in the dark was inhibited by 67±2%. Light-induced reduction of sensitivity of PEPCase to malate was decreased by cycloheximide (CHM), indicating the involvement of protein synthesis. The PEPCase kinase in protoplasts increased with time after isolation in darkness, and increased still further following light treatment. The increase in kinase activity in the light was sensitive to CHM. When protoplasts were illuminated in the presence of EGTA and the calcium ionophore A23187 to reduce intracellular Ca²⁺, the reduction in the senstivity of PEPCase to malate was enhanced, though no more PEPCase kinase activity was detected than in protoplasts illuminated in the absence of EGTA and A23187. Incubation with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) had no effect on the light-induced reduction of sensitivity of PEPCase to malate inhibition or on light-activation of PEPCase kinase. These results indicate that there is a constitutive PEPCase kinase activity in C₃ leaf tissue, that there is another kinase which is light-activated in a CHMsensitive way, that the sensitivity of PEPCase to its inhibitor may not always be correlated with apparent PEPCase kinase actvity, and that PEPCase and PEPCase kinase are regulated in a different manner in C₃ protoplasts than in C₄ protoplasts or leaf tissue.Abbreviations CAM Crassulacean acid metabolism - Chl chlorophyll - CHM cycloheximide - DCMU 3-(3,4-dichloro-phenyl)-1,1-dimethylurea - PEP phosphoenolpyruvate - PEPCase PEP carboxylase     

The anti-microtubule drug carbetamide stopsNicotiana sylvestris pollen tube growth in the style

Summary Recently, we found that the anti-microtubule drugs colchicine and propham caused the absence of microtubules and thus loss of cytoplasmic zonation in in vitro growing pollen tubes ofNicotiana sylvestris, but did not seriously affect growth. In the present study we used the herbicide carbetamide as an anti-microtubule drug. It had the same effect as colchicine and propham: the cytoplasm, including the generative cell, was no longer concentrated in the tip but was distributed randomly. In addition, ultrastructural investigations have shown that even the vesicle zone, usually found at the very tip of pollen tubes, had disappeared in some tubes. Nonetheless, in vitro growth was not inhibited by more than 20% over a period of 22 h.In contrast, tube growth in plants ceased 1 cm down in the style when carbetamide was applied to the stigma before pollination. At the lowest concentration causing this effect, microtubules of the vegetative cell had disappeared and the cytoplasm was distributed randomly, as it was for in vitro grown tubes. It can be concluded that microtubules of the vegetative cell are essential for pollen tube growth in the style.Abbreviations DAPI 4,6-diamidmo-2-phenylindole - EGTA ethyleneglycerol-bis-(aminoethyl ether) tetraacetic acid - DIC differential interference contrast - GC generative cell - IC₅₀ inhibition concentration 50% - MF microfilament - MT microtubule - PEM-buffer 50 mM PIPES 1 mM EGTA, 2 mM MgSO₄, pH 6.9 - PBS phosphate buffered saline - PIPES piperazine-bis-ethanesulphonic acid - PTG-Test pollen tube growth test - SAM substrate adhesion molecule - VC vegetative cell     

Role of Ca and Ca-activated protease in myoblast fusion

In this report, we have examined the effects of a calcium chelator, EGTA, and a calcium ionophore, A23187, on fusion of a cloned muscle cell line, L₆. Our results confirm that EGTA essentially blocks all myoblast fusion because the lateral alignment of presumptive myoblasts cannot occur in the absence of extracellular calcium. A23187, however, promotes the precocious fusion of myoblasts, apparently by facilitating Ca²⁺ transport into myoblasts. We have also demonstrated that a Ca²⁺-activated protease, CAP (mM), appears to relocate in response to the Ca²⁺ flux, changing from a random, dispersed distribution in proliferative myoblasts to a predominantly peripheral distribution in prefusion myoblasts. Coincident with the mM CAF relocation is an altered distribution of a surface glycoprotein, fibronectin. Extracellular fibronectin is seen in abundance in proliferating myoblasts, but is essentially absent from the surface of fusing myoblasts. We suggest that mM CAF when activated by Ca²⁺ influx may act to promote the release of fibronectin from the myoblast cell surface, thus providing a mechanism by which the membrane of the fusing myoblast may be rearranged to accommodate fusion.     

Are Orai1 and Orai3 channels more important than calcium influx for cell proliferation?

Transformed and tumoral cells share the characteristic of being able to proliferate even when external calcium concentration is very low. We have investigated whether Human Embryonic Kidney 293 cells, human hepatoma cell Huh-7 and HeLa cells were able to proliferate when kept 72 h in complete culture medium without external calcium. Our data showed that cell proliferation rate was similar over a range of external calcium concentration (2 μM to 1.8 mM). Incubation in the absence of external calcium for 72 h had no significant effect on endoplasmic reticulum (ER) Ca^2 + contents but resulted in a significant decrease in cytosolic free calcium concentration in all 3 cell types. Cell proliferation rates were dependent on Orai1 and Orai3 expression levels in HEK293 and HeLa cells. Silencing Orai1 or Orai3 resulted in a 50% reduction in cell proliferation rate. Flow cytometry analysis showed that Orai3 induced a small but significant increase in cell number in G₂/M phase. RO-3306, a cdk-1 inhibitor, induced a 90% arrest in G₂/M reversible in less than 15 min. Our data showed that progression through G₂/M phase after release from RO-3306-induced cell cycle arrest was slower in both Orai1 and Orai3 knock-downs. Overexpressing Orai1, Orai3 and the dominant negative non-permeant mutants E106Q-Orai1 and E81Q-Orai3 induced a 50% increase in cell proliferation rate in HEK293 cells. Our data clearly demonstrated that Orai1 and Orai3 proteins are more important than calcium influx to control cell proliferation in some cell lines and that this process is probably independent of I_CRAC and I_arc.     

On the role of calcium in chemotaxis and oscillations of dictyostelium cells

Migration of differentiated cells to a capillary containing cyclic AMP was enhanced in the presence of 1 mM CaCl2 and was virtually absent in the presence of 1 mM EGTA. Furthermore, the cells contracted and extended pseudopods to a capillary filled with the calcium ionophore A 23187. At short distances, migration to the tip of the capillary was observed, THe ionophore also induced transient decreases of the optical density of suspended cells indicating changes of cell shape. These findings support the hypothesis that cyclic AMP-binding to cell surface receptors causes a local influx of calcium ions. These in turn lead to an increase of the cytosolic calcium concentration and subsequently to an activation of cell migration. Perturbing pulses of the ionophore induced permanent phase shifts of free-running light scattering oscillations. This result indicates that cytosolic calcium is an intrinsic component of the oscillatory system.     

In-situ evidence for the involvement of calcium and bundle-sheath-derived photosynthetic metabolites in the C4 phosphoenolpyruvate-carboxylase kinase signal-transduction chain

Regulation of the light activation of C₄ phosphoenolpyruvate-carboxylase (PEPC) protein kinase (PEPC-PK) and the ensuing phosphorylation of its cytosolic target protein were studied in intact mesophyll cells (MC) and protoplasts (MP) isolated from dark-adapted leaves of Digitaria sanguinalis [L.] Scop, (hairy crabgrass). The apparent in-situ phosphorylation state of PEPC (EC 4.1.1.31) was assessed by the sensitivity of its activity in desalted MC- and MP-extracts to l-malate under suboptimal assay conditions, while the activity-state of PEPC-PK was determined by in-vitro ³²P-labeling of purified maize or recombinant sorghum PEPC by these extracts. In-situ pretreatment of intact MC at pH 8.0 by illumination and calcium addition led to significant decreases in PEPC malate sensitivity and increases in PEPC-kinase activity that were negated by the addition of EGTA to the external cell medium. Similarly, in-situ pretreatment of MP with light plus NH₄Cl at pH 7.6 led to significant decreases in malate sensitivity which did not occur when a Ca²⁺ ionophore and EGTA were included in the suspension medium. In contrast, neither EGTA nor exogenous Ca²⁺ had a major direct effect on the in-vitro activity of PEPC-PK extracted from Digitaria MC and MP. Preincubation of intact MC with 5 mM 3-phosphoglycerate or pyruvate at pH 8.0 in the dark led to significant decreases in PEPC malate sensitivity and increases in PEPC-PK activity which were not observed with various other exogenous metabolites. These collective in-situ experiments with isolated C₄ MC and MP (i) support our earlier hypothesis that alkalization of cytosolic pH is involved in the PEPC-PK signal-transduction cascade (see J.-N. Pierre et al., Eur J Biochem, 1992,210: 531–537), (ii) suggest that intracellular calcium is involved in the PEPC-kinase signal-transduction chain, but at a step upstream of PEPC-PK per se, and (iii) provide direct evidence that the bundle-sheath-derived, C₄-pathway intermediates 3-PGA and/or pyruvate also play a role in this signal-transduction cascade which ultimately effects the up-regulation of PEPC in the C₄ mesophyll cytosol.Abbreviations BS bundle-sheath - CAM Crassulacean acid metabolism - DHAP dihydroxyacetone phosphate - FPLC fast-protein liquid chromatography - Glc6P glucose 6-phosphate - I_0.5 50% inhibition constant - MC mesophyll cell(s) - MP me-sophyll protoplast(s) - PEP phosphoenolpyruvate - PEPC PEP carboxylase - PEPC-PK PEPC protein-Ser/Thr kinase - 2-PGA 2-phosphoglycerate - 3-PGA 3-phosphoglycerate - PPFD photosynthetic photon flux density - Pyr pyruvate - Ser serine The authors thank Ms. Jill Myatt for her help with some of the MC preparations. This work was supported in part by grants INT-9115566 and MCB-9315928 from the U.S. National Science Foundation (to R.C.). S.M.G.D. was a recipient of an NSERC of Canada Post-Doctoral Fellowship. This paper is Journal Series No. 11 395 of the University of Nebraska Agricultural Research Division.     

Enhanced silymarin accumulation is related to calcium deprivation in cell suspension cultures of Silybum marianum (L.) Gaertn

Elimination of calcium ions from the medium of cell cultures of Silybum marianum (L.) Gaertn increased flavonolignan production. Silymarin accumulation was not altered by treatment of cultures with the calcium ionophore A23187. The specific Ca2+ chelator, EGTA, enhanced the silymarin content in cells by 200%, and its secretion by 3-4 times. The inorganic ion La3+, as well as the calcium channel inhibitor verapamil, also stimulated production. Several reagents known to block intracellular calcium movement, such as ruthenium red, thapsigargin and TMB-8 appreciably increased silymarin accumulation. These results suggest that inhibition of external and internal calcium fluxes plays a significant role in flavonolignan metabolism of S. marianum cell cultures.     

Calcium and calcium ionophore A23187 induce high-frequency somatic embryogenesis in cultured tissues of <Emphasis Type="Italic">Coffea canephora</Emphasis> P ex Fr

High-frequency somatic embryogenesis was achieved in Coffea canephora using calcium ionophore A23187, which influences the influx of calcium into a cell. With 100 μM calcium ionophore and 5 mM calcium, 85% and 70% of cultures produced embryogenic tissue, with 105 ± 7 and 95 ± 8 primary embryos from each callus mass respectively. Medium supplemented with 100 μM EGTA (calcium chelator) or 1 mM verapamil (calcium channel blocker) significantly reduced somatic embryogenesis. Calcium imaging studies were done to determine the relationship between morphogenetic response and the cellular calcium levels. The calcium ionophore/calcium treatment was very effective in driving cellular machinery toward embryogenesis. The embryos were regenerated into plantlets when cultured on MS medium supplemented with 5 mM calcium/100 μM calcium ionophore A23187. Somatic embryogenesis-derived plants were successfully transferred to soil and grown to maturity in the field.     

Low extracellular calcium is sufficient for survival and proliferation of murine mesencephalic neural precursor cells

Various media and Ca²⁺ concentrations are employed to culture neural progenitor cells (NPCs). We have therefore explored the effects of extracellular calcium concentrations on the survival, proliferation, spontaneous apoptosis and self-renewal capacity of mesencephalic NPCs grown adherently and as free-floating neurospheres. We employed EMEM supplemented with various concentrations of extracellular CaCl₂ (0.1–1 mM). Raising the calcium concentration from 0.1 mM to 0.6 mM resulted in an increased number of NPCs growing as a monolayer and increased the protein yield of cells growing in neurospheres (24±3 μg total proteins in 0.1 mM Ca²⁺ medium vs. 316±34 μg proteins in 1 mM Ca²⁺ medium). Concentrations more than 0.6 mM did not result in a further improvement of proliferation or survival. Elimination of calcium from our control medium by 1 mM EGTA resulted in a decrease in cell number from 82±2×10⁴ NPCs/ml observed in control medium to 62±2×10⁴ NPCs/ml observed in calcium-free media. Protein yield dropped significantly in calcium-free media, accompanied by the decreased expression of the proliferation marker PCNA and the pro-survival marker Bcl-2. Two weeks of expansion as neurospheres caused spontaneous cell death in more than 90% of NPCs grown in 0.1 mM CaCl₂ EMEM compared with 42% in 1 mM CaCl₂ EMEM. Although the action of Ca²⁺ on NPCs appears to be complex, the presented data strongly suggest that extracellular calcium plays a crucial role in the maintenance of NPCs in a healthy and proliferating state; physiological concentrations (>1.0 mM) are not required, a concentration of 0.5 mM being adequate for cell maintenance.     

Calcium and A23187-induced cytolysis of mouse thymocytes

The cytotoxic effects of ionophore A₂₃₁₈₇ were studied in parallel with its action on calcium uptake in isolated mouse thymocytes. Under conditions where the cells were preincubated in a calcium-containing medium prior to ionophore treatment a close relationship could be observed between the extent of cell lysis and the stimulation of calcium uptake in the presence of A₂₃₁₈₇. In addition, increasing concentrations of calcium ions in the incubation medium lead to a pronounced decrease of cell viability and to a stimulation of calcium uptake suggesting that calcium is critical for cell survival.     

Calcium changes and the response to methyl jasmonate in rice lodicules during anthesis

Summary. Potassium pyroantimonate precipitation was used to locate loosely bound calcium in rice (Oryza sativa L.) lodicules before and after anthesis, and flowering of panicles was accelerated by treatment with methyl jasmonate. From 1 day to 4 h before anthesis, the number of calcium precipitates in the cell walls and vacuole membranes decreased gradually, whereas they increased remarkably in the cytoplasm and nucleolus. At the beginning of anthesis, the number of calcium granules in lodicules reduced sharply, but there was a large accumulation of flocculent precipitates in the vacuoles. After anthesis, the flocculent precipitates decreased in number until they disappeared, whereas the granular precipitates started to accumulate once again. The rice florets treated with 2 mM methyl jasmonate were induced to open within 10–30 min and they then closed 0.5–1 h later. The nucleolus, cytoplasm, and vacuole membrane of the lodicule cells contained many calcium granules during flowering, although the cell walls lacked calcium. At 1 h after treatment, the number of calcium granules had decreased, while flocculent precipitates were regularly observed in the nondegenerated cells. At 6 h after treatment, calcium grains started to reappear in the cell walls. These changes in calcium precipitates before and after anthesis indicate that the opening and closing of florets correlates with the calcium level in lodicule cells. In addition, excised panicles, with florets judged to be nearing anthesis, were soaked in 2–200 mM EGTA solution for 2 min after treatment with 2 mM methyl jasmonate. The results indicate that EGTA had an antagonistic effect on the methyl jasmonate-induced floret opening in rice. Correspondence and reprints: Key Laboratory of the Ministry of Education for Plant Developmental Biology, College of Life Sciences, Wuhan University, Wuhan 430072, People’s Republic of China.     

Stimulation-dependent recruitment of cytosolic phospholipase A2-alpha to EA.hy.926 endothelial cell membranes leads to calcium-independent association.

Cytosolic phospholipase A2-alpha (cPLA2-alpha) is a calcium-activated enzyme involved in agonist-induced arachidonic acid release. In endothelial cells, free arachidonic acid is predominantly converted into prostacyclin, a potent vasodilator and inhibitor of platelet activation. As the rate-limiting step in prostacyclin production is the generation of free arachidonic acid by cPLA2-alpha, this enzyme has become an attractive pharmacological target and the focus of many studies. Following stimulation with calcium-mobilizing agonists, cPLA2-alpha translocates to intracellular phospholipid membranes via its C2 domain. In this study, the calcium-induced association of cPLA2-alpha with EA.hy.926 endothelial cell membranes was investigated. Subcellular fractionation and immunofluorescence studies showed that following stimulation with histamine, thrombin or the calcium ionophore A23187, cPLA2-alpha relocated to intracellular membranes. Treatment of A23187-stimulated cells with EGTA or BAPTA-AM demonstrated that a substantial pool of cPLA2-alpha remained associated with membrane fractions in a calcium-independent manner. Furthermore, immunofluorescence microscopy studies revealed that cells stimulated for periods of greater than 10 min showed a high proportion of calcium-independent membrane-associated cPLA2-alpha. Calcium-independent membrane association of cPLA2-alpha was not due to hydrophobic or cytoskeletal interactions. Finally, the recombinant C2 domain of cPLA2-alpha exhibited calcium-independent membrane binding to membranes isolated from A23187-stimulated cells but not those isolated from nonstimulated cells. These findings suggest that novel mechanisms involving accessory proteins at the target membrane play a role in the regulation of cPLA2-alpha. Such regulatory associations could enable the cell to discriminate between the varying levels of cytosolic calcium induced by different stimuli.     

Evidence on the role of three calcium pools in Ca-ionophore A23187-stimulated rat blood platelet aggregation.

The effect of Ca-ionophore A23187 on activation of rat blood platelets was investigated to elucidate the involvement of extracellular and intracellular Ca2+ ions. Platelet aggregation induced by 10 concentrations of the stimulus was studied in Ca-free medium as well as in the presence of EGTA and/or calcium. In Ca-free medium, A23187 induced platelet aggregation in a dose-dependent way; the mean effective concentration was 1.43 +/- 0.08 mumol/l. The stimulatory effect of ionophore was potentiated by addition of 0.01 and 0.1 mM calcium and inhibited when the calcium concentration was increased to 1 mmol/l. In the presence of EGTA, A23187-stimulated aggregation of isolated rat platelets was recorded only at a 10-times higher ionophore concentration and was then reduced to 30% in comparison with aggregation in Ca-free medium. The inhibitory effect of 1 mM EGTA was abolished by addition of 2 mM calcium. We suggest the participation of at least three calcium pools in the stimulation of rat platelets by A23187, i.e. the extracellular pool, the membrane-associated pool and the pool displacing calcium intracellularly.