Leptinotoxin-h Action in Synaptosomes and Neurosecretory Cells: Stimulation of Neurotransmitter Release

Guinea pig brain cortex synaptosomes and neurosecretory PC12 cells were loaded with [3H]3,4-dihydroxyphenylethylamine ([3H]DA, [3H]dopamine) and then exposed to leptinotoxin-h (LPTx) (purified and partially purified preparations, obtained from the hemolymph of Leptinotarsa haldemani). In a Ca2+-containing Ringer medium the toxin induced prompt and massive release of the neurotransmitter. Half-maximal effects were obtained at concentrations estimated of approximately 3 X 10(-11) M for synaptosomes, and 1.5 X 10(-10) M for PC12 cells. Release responses in the two experimental systems investigated were dependent to different extents on the Ca2+ concentration in the medium. In synaptosomes clear, although slow, release of [3H]DA was elicited by the toxin even in Ca2+-free, EGTA-containing medium, provided that high (in the 10(-10) M range) concentrations were used; near-maximal responses were observed at 10(-5)M Ca2+. In contrast, the toxin-induced release from PC12 cells was appreciable only at 3 X 10(-5) M Ca2+, and was maximal at 2 X 10(-4) M and above. In both synaptosomes and PC12 cells Sr2+ and Ba2+ could substitute for Ca2+; Co2+ was inhibitory, whereas Mn2+ failed to modify the release induced by the toxin in Ca2+-containing medium. Organic blockers of the voltage-dependent Ca2+ channel (verapamil and nitrendipine) and calmodulin blocking drugs (trifluoperazine and calmidazolium) failed to inhibit the toxin-induced release of [3H]DA. LPTx induced profound morphological effects. Synaptosomes treated in the Ca2+-containing medium exhibited fusion of synaptic vesicles, formation of numerous infoldings and large cisternae, and alterations of mitochondria. In the Ca2+-free medium the effects were similar, except that their appearance was delayed, and mitochondria were well preserved. Swelling was observed in PC12 cells, accompanied by enlargement of the Golgi area, accumulation of multivesicular bodies, mitochondrial alterations, and decreased number of secretion granules (Ca2+-containing medium). Morphometric analyses revealed a good correlation between the decrease of both synaptic vesicles (synaptosomes) and neurosecretory granules (PC12 cells), and the release of [3H]DA measured biochemically. This is a good indication that the release effect of the toxin is due to stimulation of exocytosis. Taken as a whole, these results confirm the similarity of the effects of LPTx with alpha-latrotoxin of the black widow spider venom, mentioned in the companion article. However, differences in effect and target specificity suggest that the two toxins are specific to separate binding sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Further Characterization of Dopamine Release by Permeabilized PC 12 Cells

Rat pheochromocytoma cells (PC12) permeabilized with staphylococcal alpha-toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+-ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+-dependent [3H]-dopamine release from digitonin-permeabilized PC12 cells. Permeabilization with alpha-toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+-ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+-induced release. The release was unaltered by thiol-, hydroxyl-, or calmodulin-interfering substances. Thus Mg2+-ATP, calmodulin, or proteins containing -SH or -OH groups are not necessary for exocytosis in permeabilized PC12 cells.     

Leptinotoxin-h Action in Synaptosomes, Neurosecretory Cells, and Artificial Membranes: Stimulation of Ion Fluxes

Leptinotoxin-h (LPTx), a neurotoxin (otherwise designated beta-leptinotarsin-h) known to stimulate the release of neurotransmitters from synapses, was purified from the hemolymph of the potato beetle, Leptinotarsa haldemani, by a simplification of the procedure originally developed by Crosland et al. [Biochemistry 23, 734-741, (1984)]. Highly and partially purified preparations of the toxin were applied to guinea pig synaptosomes and neurosecretory (PC12) cells. When applied in a Ca2+-containing Ringer medium, at concentrations in the 10(-11) - 10(-10) M range, the toxin induced: (a) rapid depolarization of the plasma membrane, which was not inhibited by organic blockers of voltage-dependent Na+ and Ca2+ channels (tetrodotoxin or verapamil); (b) large 45Ca influx; and (c) increased free cytosolic Ca2+ concentration. These latter two effects were unaffected by verapamil. In Ca2+-free media the effects of the toxin were different in the two systems investigated. In synaptosomes, depolarization was still observed, even if the toxin concentrations needed were higher (approximately 10X) than those effective in the complete medium. In contrast, in PC12 cells no effect of the toxin on membrane potential was observed. Binding of LPTx to its cellular targets could not be investigated directly because the toxin was inactivated by the procedures used for its labeling. Indirect evidence suggested however that Ca2+ is necessary for toxin binding to PC12 cells. Interaction of LPTx with air/water interfaces, as well as with cholesterol/phospholipid mono- and bilayer membranes was investigated. The results indicate that the toxin has affinity for hydrophobic surfaces, but lacks the capacity to insert across membranes unless transpositive voltage is applied. Our results are inconsistent with the previous conclusion of Crosland et al. (1984), who suggested opening of the Ca2+ channel as the mechanism of action of LPTx. The effects of the toxin resemble those of alpha-latrotoxin (alpha-LTx) of the black widow spider venom, and therefore the two toxins might act by similar mechanisms. However, the sites recognized by the two toxins might be different, because LPTx does not inhibit alpha-LTx binding.     

Ion interaction at the pore of Lc-type Ca2+ channel is sufficient to mediate depolarization-induced exocytosis

The coupling of voltage-gated Ca2+ channel (VGCC) to exocytotic proteins suggests a regulatory function for the channel in depolarization-evoked exocytosis. To explore this possibility we have examined catecholamine secretion in PC12 and chromaffin cells. We found that replacing Ca2+ with La3+ or other lanthanide ions supported exocytosis in divalent ion-free solution. Cd2+, nifedipine, or verapamil inhibited depolarization-evoked secretion in La3+, indicating specific binding of La3+ at the pore of L-type VGCC, probably at the poly-glutamate (EEEE) locus. Lanthanide efficacy was stringently dependent on ionic radius with La3+>Ce3+>Pr3+, consistent with a size-selective binding interface of trivalent cations at the channel pore. La3+ inward currents were not detected and the highly sensitive La3+/fura-2 imaging assay (approximately 1 pm) detected no La3+ entry, cytosolic La3+ build-up, or alterations in cytosolic Ca2. These results provide strong evidence that occupancy of the pore of the channel by an impermeable cation leads to a conformational change that is transmitted to the exocytotic machinery upstream of intracellular cation build-up (intracellular Ca2+ concentration). Our model allows for a tight temporal and spatial coupling between the excitatory stimulation event and vesicle fusion. It challenges the conventional view that intracellular Ca2+ ion build-up via VGCC permeation is required to trigger secretion and establishes the VGCC as a plausible Ca2+ sensor protein in the process of neuroendocrine secretion.     

Stimulation of Calcium Uptake in PC12 Cells by the Dihydropyridine Agonist BAY K 8644

Abstract: Methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate (BAY K 8644), an analog of dihydropyridine calcium channel antagonists, stimulated ⁴⁵Ca uptake into PC12 pheochromocytoma cells. Half-maximal stimulation occurred at 80 n M BAY K 8644. Enhancement of uptake was inhibited by cationic and organic calcium channel blockers, but not by tetrodotoxin, which is consistent with an effect on voltage-dependent calcium channels. Stimulation of ⁴⁵Ca uptake by BAY K 8644 occurred only at elevated concentrations of extracellular K⁺, suggesting that BAY K 8644 may interact with calcium channels in the open (activated) state.     

Role of Ca2+ in Differentiation Mediated by Nerve Growth Factor and Dibutyryl Cyclic AMP in PC12 Cells

Abstract: Nerve growth factor (NGF) and dibutyryl cyclic AMP (dbcAMP) have synergistic effects on the neurite outgrowth of rat pheochromocytoma PC12 cells. The sites of interaction between NGF and dbcAMP have been studied extensively; however, the role of Ca²⁺ in differentiation induced by the two agents remains unclear. To understand whether intracellular Ca²⁺ is involved in the differentiation induced by the two agents, PC12 cells were treated with NGF, dbcAMP, or NGF plus dbcAMP for 2 days, and then effects on neurite outgrowth, ATP-induced Ca²⁺ influx, and Ca²⁺ mobilization from intracellular Ca²⁺ pools were examined. NGF or dbcAMP alone enhanced neurite outgrowth and Ca²⁺ accumulation by nonmitochondrial Ca²⁺ pools or the thapsigargin (TG)-sensitive Ca²⁺ pool. The dbcAMP acted synergistically with NGF to increase neurite outgrowth and to enlarge the TG-sensitive Ca²⁺ pool. The synergistic effect occurred within the first hour of treatment with dbcAMP plus NGF. On the other hand, dbcAMP abolished NGF's ability to enhance ATP-induced influx of extracellular Ca²⁺. Therefore, NGF and dbcAMP induced different effects on Ca²⁺ signaling pathways through two different but interacting pathways. In PC12 cells pretreated with TG to deplete the TG-sensitive Ca²⁺ pool, the dbcAMP- or dbcAMP plus NGF-mediated neurite outgrowth was significantly inhibited, whereas NGF-mediated neurite outgrowth was not affected by TG pretreatment. Our results suggest that the intracellular nonmitochondrial Ca²⁺ pools were changed in the differentiation process and were necessary for the synergistic effect of NGF and dbcAMP.     

Enhancement of 45Ca2+ Binding to Acidic Lipids by Barbiturates, Diphenylhydantoin, and Ethanol

Abstract: Effects of barbiturates, diphenylhydantoin, and ethanol on ⁴⁵Ca²⁺ binding to acidic lipids have been examined in an organic solvent-aqueous partition system. Hexobarbital, pentobarbital, and phenobarbital, at concentrations of 0.3 and/or 0.6 mM, enhanced the binding of ⁴⁵Ca²⁺ to phosphatidic acid, phosphatidylserine, and sulfatide but not to phosphatidylinositol or cardiolipin. Diphenylhydantoin, 0.3 mM, enhanced ⁴⁵Ca²⁺ binding to phosphatidic acid and phosphatidylserine but not to sulfatide. Ethanol at 80 mM did not enhance ⁴⁵Ca²⁺ binding to phosphatidic acid, but ethanol decreased the binding to cardiolipin and increased it to sulfatide.     

Calcium and photoperiodic flower induction in Pharbitis nil

The relationship between phytochrome-mediated induction of flowering, Ca²⁺ transport and metabolism in Pharbitis nil Chois cv. Violet seedlings has been investigated. Ethyleneglycol-bis-(β-aminoethylether)-N,N,N', N'-tetraacetic acid (EGTA), a specific Ca⁺ chelator, caused a 30–40% inhibition of flowering in Pharbitis subjected to complete photoperiodic induction. It was most effective when applied during the light period preceding along inductive dark period. The agonist of calcium channels. Bay K-8644, did not affect flowering, while Nifedipine, Verapamil and La³⁺ (antagonists of calcium channels) only slightly inhibited this process. A similar small effect has been found when the plants were treated with Li⁺ (inhibitor of the membrane phospholipids pathway), and with chlorpromazine (a camodulin inhibitor). Except for EGTA, the effect of the other substances did not depend on the timing of their application. The results of the present study suggest that the effect of all the substances applied was not specific, and flowering is not directly dependent on transport and intracellular metabolism of Ca²⁺.     

Effects of Erythropoietin on Neuronal Activity

Recently, erythropoietin (EPO) receptors and synthesis of EPO have been identified in the brain. To clarify the effects of EPO on neuronal cells, we investigated the effects of EPO on Ca2+ uptake, intracellular Ca2+ concentration, membrane potential, cell survival, release and biosynthesis of dopamine, and nitric oxide (NO) production in differentiated PC12 cells, which possess EPO receptors. EPO (10(-12)-10(-10) M) increased 45Ca2+ uptake and intracellular Ca2+ concentration in PC12 cells in a dose-related manner; these increases were inhibited by nicardipine (1 microM) or anti-EPO antibody (1:100 dilution). EPO induced membrane depolarization in PC12 cells. After a 5-day culture without serum and nerve growth factor (NGF), viable cell number decreased to 50% of that of the control cells cultured with serum and NGF. EPO (10(-13)-10(-10) M) increased the number of viable cells cultured without serum and NGF; this increase was blunted by nicardipine or anti-EPO antibody. Incubation with EPO (10(-13)-10(-10) M) stimulated mitogen-activated protein kinase activity in PC12 cells. EPO (10(-13)-10(-10) M) increased dopamine release from PC12 cells and tyrosine hydroxylase activity; these increases were sensitive to nicardipine or anti-EPO antibody. Following a 4-h incubation with EPO (10(-14)-10(-10) M), NO production was increased, which was blunted by nicardipine and anti-EPO antibody. In contrast, maximal NO synthase activity was not changed by EPO. These results suggest that EPO stimulates neuronal function and viability via activation of Ca2+ channels.     

Regulation of Calcium Concentrations in Synaptosomes: α2-Adrenoceptors Reduce Free Ca2+ by Closure of N-Type Ca2+ Channels

The relationship between intrasynaptosomal total (CaT) and free ([Ca2+]i) calcium and 45Ca accumulation was studied under physiological and K(+)-depolarised conditions in rat cortical synaptosomes. Under physiological conditions, CaT (10.7 mM) was approximately 10,000 times higher than [Ca2+]i (118 nM), showing that there is a large reservoir of sequestered calcium in synaptosomes. 45Ca accumulation was rapid (initial rate, 3.4 nmol/mg protein/min), substantial (7 nmol/mg protein in 2 min), and depolarisation dependent, and reached equilibrium after 5 min. At equilibrium, only 10% of CaT was freely exchangeable. This pool was much larger than the free Ca2+ pool. CaT, [Ca2+]i, and 45Ca accumulations were directly related to the Ca2+ concentration in the buffer, suggesting that [Ca2+]i is not highly conserved but is maintained by simple equilibria between the various pools. Clonidine reduced 45Ca accumulation in a time- and dose-dependent manner. Maximum inhibition (40% at 100 microM) occurred at 2 min and the IC50 was 80 nM. The reduction caused by clonidine (1 microM) reached equilibrium after 5 min, but this equilibrium value was lower than in controls, suggesting that clonidine changes the exchangeable Ca2+ pool size. The effects of clonidine (1 microM) on [Ca2+]i (26% reduction) and on 45Ca accumulation (24% reduction) were most apparent under physiological conditions. However, while it was not dependent on depolarisation, it did not occur in physiological buffer containing low K+ concentration (0.1-1 mM). The inhibitory effect of clonidine on 45Ca accumulation is receptor mediated as it was antagonised by idazoxan (1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

压力超负荷性心肌肥厚大鼠心肌细胞核钙转运的改变

通过腹主动脉缩窄(abdominalaorticcoarctation ,AAC)心肌肥厚大鼠模型制备、差速离心提纯心肌细胞核、酶学方法测定Ca2 +-ATPase活性、45Ca2 +同位素法测定核钙摄取和荧光分光光度计测定细胞核内自由钙浓度 ,初步揭示压力超负荷心肌肥厚大鼠心肌细胞核钙转导异常的环节。结果发现 :心肌细胞核上存在具有[Ca2 +]和ATP依赖性的高亲和力Ca2 +-ATPase ,以[Ca2 +]依赖的方式摄取45Ca2 +,并呈先升高后降低趋势。AAC术后4周大鼠心肌显著肥厚 ,伴有明显的血流动力学异常 ,与对照组比较 ,AAC大鼠心肌细胞核Ca2 +-ATPase活性减少51.93 %(p<0.001) ,但核45Ca2 +摄入量(核外[Ca2 +]浓度为800 -1600nmol/L时)和核内[Ca2 +](核外[Ca2 +]浓度为0 -1000nmol/L时)均明显增加(p<0.05) ;正常组离体心肌细胞核Ca2 +摄取受PKA刺激(p<0.05) ,而被PKC抑制剂和CaM抑制剂显著抑制(p<0.05) ,AAC大鼠心肌细胞核Ca2 +摄取仅受CaM抑制剂抑制(p<0.01) ,而PKA和PKC抑制剂对其无明显影响(p>0.05)。结论为心肌肥厚时 ,心肌细胞核Ca2 +转运系统及其磷酸化调节可能发生改变。     

糖皮质激素快速抑制高钾离子诱导嗜铬细胞瘤细胞内游离钙浓度升高 …

本研究应用钙离子特异光指示剂Ｆｕｒａ－２／ＡＭ,使用Ｍｉｒａｃａｌ影像系统检测了糖皮质激素对高钾离子升高嗜铬细胞瘤细胞（ＰＣ１２细胞）内游离钙浓度（［Ｃａ＾＋］ｉ）作用的影响。结果表明：（１）皮质酮抑制高钾离子诱导ＰＣ１２细胞［Ｃａ＾２＋］ｉ升高与其预处理细胞时间的长短有关,预处理３ｍｉｎ时,皮质酮开始产生抑制作用;预处理５ｍｉｎ时,其呈现的抑制作用最;预处理２５ｍｉｎ时,抑制作用基本消失。（２）     

Ethylene activates a plasma membrane Ca(2+)-permeable channel in tobacco suspension cells

Here, the effects of the ethylene-releasing compound, ethephon, and the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), on ionic currents across plasma membranes and on the cytosolic Ca(2+) activity ([Ca(2+)](c)) of tobacco (Nicotiana tabacum) suspension cells were characterized using a patch-clamp technique and confocal laser scanning microscopy. Exposure of tobacco protoplasts to ethephon and ACC led to activation of a plasma membrane cation channel that was permeable to Ba(2+), Mg(2+) and Ca(2+), and inhibited by La(3+), Gd(3+) and Al(3+). The ethephon- and ACC-induced Ca(2+)-permeable channel was abolished by the antagonist of ethylene perception (1-metycyclopropene) and by the inhibitor of ACC synthase (aminovinylglycin), indicating that activation of the Ca(2+)-permeable channels results from ethylene. Ethephon elicited an increase in the [Ca(2+)](c) of tobacco suspension cells, as visualized by the Ca(2+)-sensitive probe Fluo-3 and confocal microscopy. The ethephon-induced elevation of [Ca(2+)](c) was markedly inhibited by Gd(3+) and BAPTA, suggesting that an influx of Ca(2+) underlies the elevation of [Ca(2+)](c). These results indicate that an elevation of [Ca(2+)](c), resulting from activation of the plasma membrane Ca(2+)-permeable channels by ethylene, is an essential component in ethylene signaling in plants.     

Magnetic beads-assisted mild enrichment procedure for weak-binding lectins

Investigating unidentified weak-acting lectins is important for understanding glycan-related phenomena. We have developed an improved screening method for weak-acting lectins using glycan-conjugated magnetic beads (or glycobeads) involving a partial washing method and named it the mild enrichment procedure. Weak-acting lectins exist in equilibrium between bound lectin and free lectin produced by dissociation, whereas most tight-binding lectin exists in a bound state. The conventional washing step, in which the solution phase is replaced, may remove dissociated lectin from around the glycobeads; therefore, we attempted to leave a buffer space around the glycobeads to maintain the association–dissociation equilibrium of weak-acting lectins. Our results revealed that our mild enrichment procedure for screening for weak interactions, such as maltose–concanavalin A (K_a ∼ 10⁴ M⁻¹) and lactose–peanut agglutinin (K_a ∼ 10³ M⁻¹) interactions, was more effective than conventional batch methods.     

Characterization and Ca2+ Requirement of Histamine-Induced Catecholamine Secretion in Cultured Bovine Chromaffin Cells

The stimulation of cultured bovine chromaffin cells with histamine induced a continuous catecholamine secretion (EC50 = 3 x 10(-7) M) via the H1 receptor, in addition to an initial catecholamine burst due to a nonspecific stimulatory effect at higher doses (greater than or equal to 10(-4) M). The continuous secretion showed little desensitization and lasted for more than 1 h. In fura-2-loaded cells, the stimulation with histamine evoked a transient rise of intracellular free Ca2+ concentration ([Ca2+]i) which lasted only for a few minutes and was followed by a sustained [Ca2+]i rise which continued for more than 20 min. The addition of an activator for the L-type voltage-sensitive Ca2+ channel, i.e., Bay K 8644 (1 microM), facilitated the sustained [Ca2+]i rise, as well as the secretion, whereas the addition of relatively high concentrations of Ca(2+)-channel blockers (10 microM) suppressed the sustained [Ca2+]i rise and part of the secretion. Removal of extracellular Ca2+ completely abolished continuous secretion and sustained [Ca2+]i rise. When the external Ca2+ level was elevated, both sustained [Ca2+]i rise and continuous secretion were enhanced in a similar Ca(2+)-dependent manner, showing saturation with around 1-3 mM Ca2+. This Ca2+ dependence was clearly different from that observed with high K+ and nicotine, which is mediated by the L-type Ca2+ channel, in which the responses showed little or no saturation when the Ca2+ level was increased. The results indicate that stimulation with histamine induces a continuous secretion via the H1 receptor, in addition to a transient and nonspecific secretion at higher doses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Substance P on Carbachol-Stimulated 45Ca2+ Uptake into Cultured Adrenal Chromaffin Cells

Substance P is known to modulate acetylcholine-induced catecholamine release from adrenal chromaffin cells. To investigate the mechanisms involved in this modulation, the present study examined the effects of substance P on net 45Ca2+ fluxes in cultures of bovine adrenal chromaffin cells. Two effects of substance P were observed: (1) Substance P inhibited carbachol-induced 45Ca2+ uptake and 45Ca2+ efflux and (2) substance P protected against desensitization of carbachol-induced 45Ca2+ uptake and 45Ca2+ efflux. Thus substance P modulates two other cholinergic responses, 45Ca2+ uptake and 45Ca2+ efflux, in a manner similar to its modulation of catecholamine release. The results also indicate that substance P's inhibition of net carbachol-induced 45Ca2+ uptake is due to inhibition of 45Ca2+ uptake rather than enhancement of 45Ca2+ efflux. Substance P almost completely inhibited carbachol-induced 45Ca2+ uptake in both Na+-containing and Na+-free media, suggesting that substance P can inhibit the uptake of 45Ca2+ induced by carbachol regardless of whether 45Ca2+ is taken up through voltage-sensitive or acetylcholine receptor-linked channels. However, substance P produced only a small inhibition of K+-induced 45Ca2+ uptake, indicating that substance P does not interact directly with voltage-sensitive Ca2+ channels. In addition, substance P's inhibition of carbachol-induced 45Ca2+ uptake was noncompetitive with respect to Ca2+, were unable to overcome substance P's inhibition of [3H]-norepinephrine ( [3H]NE) release. It is concluded that substance P does not interact directly with Ca2+ channels in bovine adrenal chromaffin cells.     

A study on concanavalin a binding to human erythrocytes

Interactions of concanavalin A with human erythrocytes were studied using ¹²⁵I-labelled concanavalin A and a centrifugal technique with dibutyl phthalate which permitted complete separation of bound and free concanavalin A. Binding of ¹²⁵I-labelled concanavalin A to human erythrocytes was dependent on cell concentration, pH and temperature. Specificity of binding was confirmed by inhibition and dissociation studies with sugars and native concanavalin A. Positive cooperative binding of concanavalin A to human erythrocytes was observed at low concanavalin A concentrations (less than 1 μ/ml) in both buffers studied. Positive cooperativity at higher concanavalin A concentrations (more than 100 μ/ml) was seen in Tris-Hepes buffer but not in phosphate-buffered saline. Consistent with this cooperative effect was the observation that although dissociation of ¹²⁵I-labelled concanavalin A from the erythrocytes was complete in the presence of 1 mg/ml of the native lectin, release was inhibited by low concentrations (1 μ/ml). A comparison of concanavalin A binding with hemagglutination studies suggest that the amount of concanavalin A bound determines the rate of erythrocyte agglutination and the size of the aggregates formed.