培养牛心内膜内皮细胞的腺苷三磷酸双磷酸酶的特性研究

包括血管内皮细胞在内的多种细胞和组织存在腺苷三磷酸双磷酸酶,但心内膜内皮细胞是否含有ａｐｙｒａｓｅ尚无报道。本文旨在研究牛心内膜内皮细胞ａｐｙｒａｓｅ的特性。以无机磷释放法检测培养牛心内膜内皮细胞（ＢＥＥＣ）ａｐｙｒａｓｅ的活性,公认的ａｐｙｒａｓｅ抑制剂叠氮钠呈浓度依赖性地抑制ａｐｙｒａｓｅ活性;另一种ａｐｙｒａｓｅ抑制剂氟化钠也明显抑制ａｐｙｒａｓｅ活性地抑制活性而Ｎａ＾＋／Ｋ＾＋－ＡＴＰａｓ     

N-acetylcysteine-induced vasodilation involves voltage-gated potassium channels in rat aorta

AimsN-acetylcysteine (NAC) has a protective effect against vascular dysfunction by decreasing the level of reactive oxygen species (ROS) in experimental and human hypertension. This study was designed to examine whether NAC would relax vascular rings in vitro via nitric oxide–cyclic guanosine monophosphate (NO–cGMP) pathway, extracellular Ca²⁺ and/or K⁺ channels.Main methodsRat aortic arteries were mounted in an organ bath, contracted with 0.1, 0.5 or 1 µmol/L phenylephrine to plateau, and the vasodilatory effect of NAC was examined in the absence or presence of ROS scavengers, inhibitors of NO–cGMP pathway or K⁺ channels. Vascular smooth muscle cells (VSMCs) were loaded with a calcium sensitive fluorescent dye fluo-3 AM, and [Ca²⁺]_i was determined with laser-scanning confocal microscopy.Key findingsNAC (0.1–4 mmol/L) dose-dependently relaxed rat aorta pre-contracted with phenylephrine. Endothelium removal, endothelial nitric oxide synthase inhibitor N^ω-Nitro-l-arginine (L-NNA) (100 µmol/L) or soluble guanylyl cyclase (sGC) inhibitor (ODQ) (10 µmol/L) did not affect NAC-induced vasodilation. In contrast, NAC-induced vasodilation was blunted after extracellular calcium was removed and calcium imaging showed that 4 mmol/L NAC quickly decreased [Ca²⁺]_i in fluo-3 AM loaded VSMCs. NAC-induced vasodilation was significantly reduced in the presence of voltage-gated K⁺ channels (Kv) inhibitor 4-aminopyridine (4-AP).SignificanceThe vasodilatory effect of NAC may be explained at least partly by activation of voltage-gated K⁺ channels.     

Nitric oxide induces transient Ca2+ changes in endothelial cells independent of cGMP

Ca²⁺ changes induced by nitric oxide (NO^·) were investigated in cultured human endothelial cells. Sodium nitroprusside (SNP) (1–100 μmol/L) and S-Nitroso-N-acetylpenicillamine (SNAP) (100 μmol/L) were used as NO^· donors. The cytoplasmatic Ca²⁺ concentration was calculated using ratiometric FURA2 fluorescence measurements. Both NO^· donors caused transient oscillatory Ca²⁺ changes, which were not detectable in the presence of oxyhemoglobin (50 μmol/L). Digital ratio imaging revealed initiation sites within cells where Ca²⁺ increases started spreading, which indicates that nonuniformly distributed targets might be involved in these reactions. Calcium was released from intracellular stores as indicated by experiments performed in Ca²⁺-free buffer. L-type Ca²⁺-channel blocker diltiazem (100 μmol/L) was not able to block these responses. NO^·-induced Ca²⁺ release from intracellular stores caused capacitative Ca²⁺ entry. Both thapsigargin (1 μmol/L) and cyclopiazonic acid (10 μmol/L) inhibited the SNP response completely, whereas neither ryanodine (up to 100 μmol/L) nor dantrolene (100 μmol/L) was able to inhibit Ca²⁺ changes induced by SNP, indicating that primarily inositol 1,4,5-triphosphate (IP₃)-dependent stores are released upon stimulation with NO^·. A small inhibitory effect of ATP- and SNP-induced peak [Ca²⁺]_i increase was measured in the presence of both caffeine (20 mmol/L) and procaine (1 mmol/L). Evidence is presented that cGMP is not involved in NO^·-induced Ca²⁺ signals, as neither inhibitors of guanylate cyclase (methylene blue and LY (83583) nor cell permeant analogues of cGMP altered or simulated [Ca²⁺]_i changes. An inhibitor of cGMP-dependent protein kinase was also ineffective. We therefore propose that endothelial cells have specific targets proximal or at IP₃ receptors to induce Ca²⁺ changes in endothelial cells stimulated with NO^·. J. Cell. Physiol. 172:296–305, 1997. © 1997 Wiley-Liss, Inc.     

Role of calcium mobilization in the regulation of spontaneous transient outward currents in porcine coronary artery myocytes

The purpose of the present study was to further study the characteristics and regulation of spontaneous transient outward currents (STOCs) in freshly isolated porcine coronary artery smooth muscle cells (ASMCs). STOCs were recorded using the perforated whole-cell patch-clamp configuration. STOCs were voltage-dependent and superimposed stochastically onto whole-cell Ca²⁺-activated-K⁺ (BK_Ca) currents. Charybdotoxin (ChTX, 200 nmol/L), a selective blocker of BK_Ca channels, completely inhibited STOCs within 10 min. STOCs activity was greatly suppressed when extracellular Ca²⁺ concentration decreased from 1.8 mmol/L to 200 nmol/L, further removal of Ca²⁺ abolished STOCs activity. Ca²⁺ ionophore A23187 (10 μmol/L) increased STOCs activity significantly. Verapamil (20 μmol/L) and CdCl₂ (200 μmol/L), two kinds of organic L-type voltage-dependent Ca²⁺ channels (L-VDCCs) antagonists, had little effect on STOCs. In addition, the ryanodine receptors (RyRs) agonist caffeine (5 mmol/L) significantly activated STOCs. Application of ryanodine (50 μmol/L) to block RyRs abolished STOCs, subsequent washout of ryanodine or application of caffeine failed to reproduce STOCs activity. Inhibition of inositol 1,4,5-trisphosphate receptors (IP₃Rs) by 2APB (40 μmol/L) greatly suppressed the activity of STOCs, application of caffeine (5 mmol/L) in the presence of 2APB caused a burst of outward currents followed by inhibition of STOCs. These results suggest that STOCs in porcine coronary ASMCs are mediated by BK_Ca channels. Extracellular Ca²⁺ is essential for STOCs activity, while Ca²⁺ entry through L-VDCCs has little effect on STOCs. Intracellular Ca²⁺ release induced by RyRs is responsible for the regulation of STOCs, whereas IP3Rs might also be involved.     

Identification and characterization of an anti-fungi Fusarium oxysporum f. sp. cucumerium protease from the Bacillus subtilis strain N7

A newly discovered alkaline antifungal protease named P6 from Bacillus subtilis N7 was purified and partially characterized. B. subtilis N7 culture filtrates were purified by 30–60% (NH₄)₂SO₄ precipitation, anion-exchange chromatography and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single band of 41.38 kDa. Peptide sequence of protease P6 was determined using a 4800 Plus MALDI TOF/TOF? Analyzer System. Self-Formed Adaptor PCR (SEFA-PCR) was used to amplify the 1,149 bp open read frame of P6. Dimensional structure prediction using Automatic Modeling Mode software showed that the protease P6 consisted of two β-barrel domains. Purified P6 strongly inhibited spore and mycelium growth of Fusarium oxysporum f. sp. cucumerium (FOC) by causing hypha lysis when the concentration was 25 μg/ml. Characterization of the purified protease indicated that it had substrate specificity for gelatin and was highly active at pH 8.0–10.6 and 70°C. The P6 protease was inhibited by EDTA (2 mmol/L), phenyl methyl sulfonyl fluoride (PMSF, 1 mmol/L), Na⁺, Fe³⁺, Cu²⁺, Mg²⁺ (5 mmol/L each) and H₂O₂ (2%, v/v). However, protease activity was activated by Ca²⁺, K⁺, Mn²⁺ (5 mmol/L each), mercaptoethanol (2%, v/v) and Tween 80 (1%, v/v). In additon, activity was also affected by organic solvents such as acetone, normal butanol and ethanol, but not hexane (25%, v/v each).     

Role of calcium mobilization in the regulation of spontaneous transient outward currents in porcine coronary artery myocytes

The purpose of the present study was to further study the characteristics and regulation of spontaneous transient outward currents (STOCs) in freshly isolated porcine coronary artery smooth muscle cells (ASMCs). STOCs were recorded using the perforated whole-cell patch-clamp configuration. STOCs were voltage-dependent and superimposed stochastically onto whole-cell Ca2 -activated-K (BKCa) currents. Charybdotoxin (ChTX, 200 nmol/L), a selective blocker of BKCa channels, completely inhibited STOCs within 10 min. STOCs activity was greatly suppressed when extracellular Ca2 concentration decreased from 1.8 mmol/L to 200 nmol/L, further removal of Ca2 abolished STOCs activity. Ca2 ionophore A23187 (10 μmol/L) increased STOCs activity significantly. Verapamil (20 μmol/L) and CdCl2 (200 μmol/L), two kinds of organic L-type voltage-dependent Ca2 channels (L-VDCCs) antagonists, had little effect on STOCs. In addition, the ryanodine receptors (RyRs) agonist caffeine (5 mmol/L) significantly activated STOCs. Application of ryanodine (50 μmol/L) to block RyRs abolished STOCs, subsequent washout of ryanodine or application of caffeine failed to reproduce STOCs activity. Inhibition of inositol 1,4,5-trisphosphate receptors (IP3Rs) by 2APB (40 μmol/L) greatly suppressed the activity of STOCs, application of caffeine (5 mmol/L) in the presence of 2APB caused a burst of outward currents followed by inhibition of STOCs. These results suggest that STOCs in porcine coronary ASMCs are mediated by BKCa channels. Extracellular Ca2 is essential for STOCs activity, while Ca2 entry through L-VDCCs has little effect on STOCs. Intracellular Ca2 release induced by RyRs is responsible for the regulation of STOCs, whereas IP3Rs might also be involved.     

Extracellular ATP Released by Osteoblasts Is A Key Local Inhibitor of Bone Mineralisation

Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PP_i). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2P_i). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PP_i-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation.     

不同浓度Fe2+与Zn2+对羊草幼苗生长和生理特性的影响

以羊草幼苗为研究对象,通过调整全营养培养基(CK,0.05 mmol/L Fe²⁺、0.015 mmol/L Zn²⁺)中铁或者锌含量设置0、10倍、20倍Fe²⁺(Zn²⁺)浓度处理Fe₀(Zn₀)、Fe₁₀(Zn₁₀)、Fe₂₀(Zn₂₀),以及在高铁培养基中单独添加0.15 mmol/L Zn²⁺或同时添加10 mmol/L Ca²⁺、5 mmol/L Mg²⁺、20 mmol/L K⁺处理,测定培养6 d后幼苗生长指标和矿质元素含量、以及高铁(Fe₂₀)处理下幼苗根中抗氧化指标和相关基因表达量,探究不同浓度Fe²⁺、Zn²⁺对羊草幼苗生长、矿质元素吸收积累及抗氧化指标、基因表达的影响。结果表明：(1)缺锌(Zn₀)显著抑制羊草幼苗鲜重的增加和Zn元素的积累,但促进Fe、Mg元素的积累;高浓度锌(Zn₁₀、Zn₂₀)显著促进幼苗叶片生长和Zn元素的积累;缺铁(Fe₀)显著抑制幼苗的根长、鲜重和Fe元素的积累,促进Mg、Zn元素的积累;高浓度铁(Fe₁₀、Fe₂₀)显著抑制羊草幼苗根叶生长、根毛发育和Ca、Zn、Mg、K元素的积累。(2)增加Zn²⁺和Ca²⁺、Mg²⁺、K⁺浓度无法恢复高铁胁迫对幼苗生长的抑制作用。(3)高浓度铁(Fe₂₀)处理羊草幼苗48 h后,根部过氧化物酶、超氧化物歧化酶、过氧化氢酶、抗坏血酸过氧化物酶、谷胱甘肽还原酶活性和丙二醛、抗坏血酸、还原型谷胱甘肽含量显著升高;烟酰胺合成酶基因、过氧化物酶基因表达量显著下调,植物类萌发素蛋白基因表达量显著上调。研究发现,羊草幼苗生长发育和矿质元素积累对环境中Zn²⁺浓度变化不敏感,却受到环境中高浓度Fe²⁺的显著抑制,并造成严重的氧化胁迫伤害,这种伤害无法在添加Zn²⁺或同时添加Ca²⁺、Mg²⁺、K⁺的条件下恢复。     

Properties of apyrase and inorganic pyrophosphatase inStreptomyces aureofaciens

Apyrase (ATP-diphosphohydrolase, EC 3.6.1.5) and inorganic pyrophosphatase (EC 3.6.1.1) were partially purified fromS. aureofaciens RIA 57 and characterized. Apyrase degrades, in addition to ATP, other nucleoside triphosphates and nucleoside diphosphates, diphosphate, thiamine diphosphate, phosphoenolpyruvate and oligophosphates of chain lengthn ≦ 90. The apyrase activity was detected in the membrane and supernatant fractions. Its properties (substrate specificity, effect of inhibitors, pH optimum and effect of Mg²⁺ ions) were similar in both fractions except for the effect of oligomycin that inhibited only the membrane fraction. Pyrophosphatase exhibited a strict substrate specificity, substrates other than diphosphate being degraded relatively slowly. Of other enzymes exhibiting the phosphatase activity acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1), trimetaphosphatase (EC 3.6.1.2) and exopolyphosphatase (EC 3.6.1.11) degrading oligophosphates of chain lengthn = 15, 40 and 60, were detected.     

Enhancing Effect of Zinc on <Emphasis Type="SmallCaps">l</Emphasis>-Histidine Transport in Rat Lung Microvascular Endothelial Cells

The aim of this study was to examine enhancing effect of l-histidine into cultured rat lung microvascular endothelial cells (LMECs), which constitute the gas–blood barrier. Uptake of l-histidine into LMECs markedly increased with the addition of ZnSO₄ (0.1 mmol/L), and this enhanced uptake of l-histidine was drastically reduced in the presence of the Na⁺-independent system L substrate, 2-amino-2-norbornanecarboxylic acid (BCH). However, the uptake of l-histidine together with ZnSO₄ was not reduced by the addition of metabolic inhibitor, 2,4-dinitrophenol, or sodium ion replacement. Moreover, the addition of the system N-substrate, l-glutamic acid γ-monohydroxamate did not significantly decrease the uptake of l-histidine with 143 mmol/L Na ⁺ + 1 mmol/L BCH. These results indicated that system-N transporter does not play a role in the uptake of l-histidine in the presence of ZnSO₄, suggesting that only system-L transporter is involved in the uptake of l-histidine, although l-histidine in the absence of ZnSO₄ was taken up by at least two pathways of Na⁺-dependent system-N and Na⁺-independent system-L processes into rat LMECs. The uptake of l-histidine into rat LMECs in the presence of ZnSO₄ was also found to be unaffected by pH (5.0–7.4), indicating that uptake of l-histidine into LMECs by the addition of zinc may not be involved in the H⁺-coupled transporters.     

Tonoplast inorganic pyrophosphatase in Vicia faba guard cells

Lysed guard-cell protoplasts of Vicia faba L. exhibited hydrolytic activity characteristic of tonoplast inorganic pyrophosphatase (V-PPase; EC 3.6.1.1). Activity was inhibited by the specific V-PPase inhibitor aminomethylenediphosphonate, stimulated by K⁺ (K _m = 51 mM) and inhibited by Ca²⁺ (80 nM free Ca²⁺ was required for 50% inhibition at 0.27 mM free Mg²⁺). Patch-clamp measurements of electrogenic activity confirmed enzyme localisation at the tonoplast. This is the first report of V-PPase activity in guard cells; its possible involvement in stomatal opening is discussed. Received: 12 February 1998 / Accepted: 24 April 1998     

ATP Released by Electrical Stimuli Elicits Calcium Transients and Gene Expression in Skeletal Muscle

ATP released from cells is known to activate plasma membrane P2X (ionotropic) or P2Y (metabotropic) receptors. In skeletal muscle cells, depolarizing stimuli induce both a fast calcium signal associated with contraction and a slow signal that regulates gene expression. Here we show that nucleotides released to the extracellular medium by electrical stimulation are partly involved in the fast component and are largely responsible for the slow signals. In rat skeletal myotubes, a tetanic stimulus (45 Hz, 400 1-ms pulses) rapidly increased extracellular levels of ATP, ADP, and AMP after 15 s to 3 min. Exogenous ATP induced an increase in intracellular free Ca²⁺ concentration, with an EC₅₀ value of 7.8 ± 3.1 μm. Exogenous ADP, UTP, and UDP also promoted calcium transients. Both fast and slow calcium signals evoked by tetanic stimulation were inhibited by either 100 μm suramin or 2 units/ml apyrase. Apyrase also reduced fast and slow calcium signals evoked by tetanus (45 Hz, 400 0.3-ms pulses) in isolated mouse adult skeletal fibers. A likely candidate for the ATP release pathway is the pannexin-1 hemichannel; its blockers inhibited both calcium transients and ATP release. The dihydropyridine receptor co-precipitated with both the P2Y₂ receptor and pannexin-1. As reported previously for electrical stimulation, 500 μm ATP significantly increased mRNA expression for both c-fos and interleukin 6. Our results suggest that nucleotides released during skeletal muscle activity through pannexin-1 hemichannels act through P2X and P2Y receptors to modulate both Ca²⁺ homeostasis and muscle physiology.     

Acrylamide-induced effects on general and neurospecific cellular functions during exposure and recovery

Basal cytotoxicity, morphological changes and alterations in cell physiological and neurochemical functions were studied in differentiated human neuroblastoma (SH-SY5Y) cells during exposure to acrylamide and during a subsequent recovery period after cessation of exposure. Acrylamide induced a 20% reduction in the number of neurites per cell at 0.21 mmol/L and 20% decrease in the protein synthesis rate at 0.17 mmol/L after 72 h of exposure. Furthermore, the basal level of intracellular calcium concentration ([Ca²⁺]_i) and receptor-activated (carbachol, 0.1 mmol/L) Ca²⁺ fluxes increased by 49% and 21%, respectively, at 0.25 mmol/L. These observations were made at noncytotoxic acrylamide concentrations, signifying specific neurotoxic alterations. Forty-eight hours after cessation of acrylamide exposure, the SH-SY5Y cells had recovered, i.e., the number of neurites per cell as well as the basal level of [Ca²⁺]_i and rate of protein synthesis were comparable to those of control cells. The general calpain inhibitor calpeptin decreased the acrylamide-induced (0.5 mmol/L) neurite degeneration, determined as reduction in number of neurites per cell, from 52% to 17% as compared to control cells, which further supports the hypothesis that an increased [Ca²⁺]_i plays a significant role for acrylamide-induced axonopathy.     

Na+/K+/Cl− cotransport in cultured vascular smooth muscle cells: Stimulation by angiotensin II and calcium ionophores,inhibition by cyclic AMP and calmodulin antagonists

Summary The specific activity of the Na⁺/K⁺/Cl^– cotransporter was assayed by measuring the initial rates of furosemide-inhibitable⁸⁶Rb⁺ influx and efflux. The presence of all three ions in the external medium was essential for cotransport activity. In cultured smooth muscle cells furosemide and bumetanide inhibited influx by 50% at 5 and 0.2 m, respectively. The dependence of furosemide-inhibitable⁸⁶Rb⁺ influx on external Na⁺ and K⁺ was hyperbolic with apparentK _m values of 46 and 4mm, respectively. The dependence on Cl^– was sigmoidal. Assuming a stoichiometry of 112 for Na⁺/K⁺/Cl^–, aK _m of 78mm was obtained for Cl^–. In quiescent smooth muscle cells cotransport activity was approximately equal to Na⁺ pump activity with each pathway accounting for 30% of total⁸⁶Rb⁺ influx. Growing muscle cells had approximately 3 times higher cotransport activity than quiescent ones. Na⁺ pump activity was not significantly different in the gorwing and quiescent cultures. Angiotensin II (ANG) stimulated cotransport activity as did two calcium-transporting ionophores, A23187 and ionomycin. The removal of external Ca²⁺ prevented A23187, but not ANG, from stimulating the cotransporter. Calmodulin antagonists selectively inhibited⁸⁶Rb⁺ influx via the cotransporter. Beta-adrenoreceptor stimulation with isoproterenol, like other treatments which increase cAMP, inhibited cotransport activity. Cultured porcine endothelial cells had 3 times higher cotransport activity than growing muscle cells. Calmodulin antagonists inhibited cotransport activity, but agents which increase cAMP or calcium had no effect on cotransport activity in the endothelial cells.     

The effect of boromycin on the Ca2+ homeostasis

A boron-containing antibiotic, boromycin (BM), was found to influence the Ca²⁺ homeostasis in both excitable and non-excitable cells. In non-excitable cells (human erythrocytes and leucocytes) it inhibited the resting passive⁴⁵Ca²⁺ transport in 10^–6–10^–5 mol/L concentrations. In human erythrocytes, the passive ⁴⁵Ca²⁺ transport induced by the presence of 1 mmol/L NaVO₃ was inhibited by boromycin (90% inhibition) as well. The inhibitory effect of BM on the NaVO₃-induced passive ⁴⁵Ca²⁺ transport was diminished in the presence of inhibitory concentrations of nifedipine (10 mol/L – 60% inhibition) or of those of K⁺ _o (75 mmol/L – 20% inhibition). On the other hand, in rat brain synaptosomes, and rat cardiomyocytes, BM stimulated the passive ⁴⁵Ca²⁺ transport in resting cells at similar concentrations. In rat cardiomyocytes the stimulation was transient. The stimulatory effect on the passive ⁴⁵Ca²⁺ transport in rat brain synaptosomes was accompanied with the increase of cytoplasmic Ca²⁺ concentration measured by means of the entrapped fluorescent Ca²⁺ chelator fura-2. The stimulatory effect of BM was diminished when synaptosomes were pre-treated with veratridine (10 mol/L) which itself stimulated the passive ⁴⁵Ca²⁺ transport. At saturating concentrations of veratridine, no stimulatory effect of BM was observed. These results could be explained by the indirect interaction of BM with both Ca²⁺ and Na⁺ transport systems via transmembrane ionic gradients of monovalent cations and could be useful in determining whether the cells belong to excitable, or non-excitable cells.     

Regulation of the Na+-K+(NH)-2Cl− cotransporter of rat submandibular glands

A cellular suspension from rat submandibular glands was exposed to different concentrations of NH₄Cl, and the variations of the intracellular concentration of calcium ([Ca²⁺]_i) and the intracellular pH (pH_i) were measured using fura-2 and 2′,7′-bis-(2-carboxy-ethyl)-5(6)-carboxyfluorescein. More than 5 mmol/l NH₄Cl significantly increased the [Ca²⁺]_i without affecting the response to 100 µmol/l carbachol. When exposed to 1 and 5 mmol/l NH₄Cl, the cells acidified immediately. At 30 mmol/l, NH₄Cl first alkalinized the cells and the pH_i subsequently dropped. This drop reflects the uptake of NH ions that dissociate to NH₃ and H⁺ in the cytosol. These protons are exchanged for extracellular sodium by the Na⁺/H⁺ exchanger because the presence of an inhibitor of the exchanger in the medium increased the acidification induced by 1 mmol/l NH₄Cl. Ouabain partly blocked the uptake of NH. In the combined presence of ouabain and bumetanide (an inhibitor of the Na⁺-K⁺-2Cl⁻ cotransporter), 1 mmol/l NH₄Cl alkalinized the cells. The contribution of the Na/K ATPase and the Na⁺-K⁺-2Cl⁻ cotransporter in the uptake of NH was independent of the presence of calcium in the medium. Isoproterenol increased the uptake of NH by the cotransporter. Conversely, 1 mmol/l extracellular ATP blocked the basal uptake of NH by the cotransporter. This inhibition was reversed by extracellular magnesium or Coomassie Blue. It was mimicked by benzoyl-ATP but not by CTP, GTP, UTP, ADP, or ADPβS. ATP only slightly inhibited the increase of cyclic AMP (−22%) by isoproterenol but fully blocked the stimulation of the cotransporter by the β-adrenergic agonist. ATP increased the release of ³H-arachidonic acid from prelabeled cells but SK&F 96365, an imidazole-based cytochrome P450 inhibitor, did not affect the inhibition by ATP. It is concluded that the activation of a purinoceptor inhibits the basal and the cyclic AMP-stimulated activity of the Na⁺-K⁺-2Cl⁻ cotransporter. J. Cell. Physiol. 180:422–430, 1999. © 1999 Wiley-Liss, Inc.     

NaCl胁迫下沙枣幼苗生长和阳离子吸收、运输与分配特性

沙枣(Elaeagnus angustifolia L.)耐盐性强,是我国北方生态脆弱地区造林绿化的一个先锋树种。为探讨沙枣的盐适应机制,研究了不同浓度NaCl(0、100和200 mmol/L)胁迫30d对其水培幼苗生物量累积以及不同组织(根、茎、叶)K+、Na+、Ca2+和Mg2+吸收、运输与分配的影响。结果表明:盐胁迫不同程度地促进了沙枣苗根系生长;100 mmol/L NaCl胁迫对幼苗生物量累积无明显影响,而200 mmol/L则显著抑制了生物量累积;盐胁迫幼苗根、茎、叶中Na+含量以及K+-Na+选择性运输系数(S K,Na)和Ca2+-Na+选择性运输系数(S Ca,Na)显著或大幅度增加,而K+、Ca2+、Mg2+含量以及K+/Na+、Ca2+/Na+和Mg2+/Na+比值则显著或大幅度下降;200 mmol/L NaCl胁迫沙枣根Na+含量和根Na+净累积量分别为22.15 mg/g干重和1.87 mg/株(是对照的16.20倍和20.06倍),根成为Na+净累积量增加幅度最大的组织和Na+含量最高的组织;200 mmol/L NaCl胁迫沙枣茎、叶中的Na+含量以及冠组织Na+净累积量分别高达5.15、7.71 mg/g干重和3.29 mg/株(是对照的7.22倍、9.58倍和5.45倍),但幼苗仍能正常生长。综合分析认为,沙枣的盐适应机制是根系拒盐和冠组织耐盐,主要通过根系的补偿生长效应、根系对Na+的聚积与限制作用以及冠组织对Na+的忍耐来实现的,同时也与根、茎和叶对K+、Ca2+选择性运输能力显著增强有关。     

Differential effects of ca2+ and Mg2+ on endonuclease activation in isolated promyelocytic HL-60 cell nuclei

In autodigestion assays, endonucleaw activity in non-apoptotic HL-60 promydocytic leukemia cell nuclei cleaved the chromatin of he autologous cells to an oligonucleosomal length pattern. Both EGTA and EDTA inhibited the activation of endonuclease activity in isolated HL-60 cell nuclei. The inhibition by EDTA could be reversed by exogenous Ca²⁺. but not by exogenous Mg²⁺. In Ca²⁺/Mg²⁺-free nuclei digation buffer, addition of Ca^2→ (1-10 mmol/L) induced endonuclease activity in the isolated nuclei, while addition of Mg²⁺ had no effect. In the presence of Ca²⁺(0.1 mmol/L), endonuclease activity was enhanced by exogenous Mg²⁺ (0.1-10mmol/L). These results suggest that the endonuclease responsible for internucleosomal DNA fragmentation in HL-60 cells during apoptosis is activated by Ca²⁺ and further modulated by Mg²⁺ in the presence of ca²⁺.     

Partial purification and some properties of urease from the alkaliphilic cyanobacteriumNostoc calcicola

Urease extracted from an alkaliphilic diazotrophic cyanobacteriumNostoc calcicola was partially purified and some of its properties were studied. Urease purified 39-fold from the crude enzyme extract showed its optimum activity at pH 7.5 and at 40°C with aK _m value of 120 μmol/L. The enzyme was found to be sensitive to metal cations, particularly Hg²⁺, Ag⁺ and Cu²⁺. 4-Hydroxymercuribenzoate (a mercapto-group inhibitor) and acetohydroxamic acid (a chelating agent of nickel) inhibited, the enzyme activity completely. These results suggest the involvement of an SH-group and Ni²⁺ in the activity of urease fromN. calcicola.     

Cobalt ions enhance light responsiveness of carp cone horizontal cells in low calcium

Effects of cobalt ions (Co2+) on horizontal cells in low extracellular calcium were examined in isolated, superfused carp retinas. While 0.1mmol/L Co2+ completely suppressed both rod- and cone-driven horizontal cells in normal Ringer's solution, it enhanced light responses of cone horizontal cells in low (0.1mmol/L) calcium. The enhancement of the cone horizontal cell response by Co2+ was not caused by changes in light responsiveness of cone photoreceptors. Moreover, application of 50μmol/L IBMX, an inhibitor of phosphodiester enzyme, reduced the suppressive effect of 0.1 mmol/L Co2+ in normal Ringer's solution. In consequence, the above-described enhancement of the cone horizontal cell light responsiveness may be due to a depolarization of cones caused by low calcium, which increases the activity of voltage-dependent calcium channels at cone terminals.