ATP-induced [Ca2+]i changes in the human corneal epithelium

ATP, when leaked from damaged cells, is capable of eliciting responses in neighboring cells. A better understanding of the mechanism of this response is essential for designing therapeutic strategies for disease, there have been only a limited number of studies on the effect of ATP on the human cornea. We examined ATP-induced intracellular Ca2+ ([Ca2+]i) changes in the human corneal epithelia, cultured to near confluence. Cells were loaded with the Ca2+ indicators, Indo-1 or Fluo-4, and [Ca2+]i was monitored. ATP was found to induce an increase in [Ca2+]i, which was initiated from the perinuclear region and the nuclear envelope per se, and then propagated gradually towards the periphery. Intranuclear Ca2+ was momentarily increased. UTP elicited an identical response, but adenosine and alpha, beta-methylene ATP had no effect. Pretreatment with U73122 or thapsigargin inhibited the ATP-induced increase in [Ca2+]i. When a cell was topically stimulated with ATP, the [Ca2+]i increase spread beyond the cell boundary. The intercellular communications that accompanied the [Ca2+]i changes were inhibited by octanol. We conclude that extracellular ATP in the human cornea caused the mobilization of Ca2+ from intracellular Ca2+ stores (e.g. the endoplasmic reticulum and nuclear envelope) via P2Y purinoceptors of the epithelial cell. The response to ATP appears to spread to neighboring regions through gap junctions in the epithelium.     

Resting [Ca2+]i and [Ca2+]i transients are similar in fibroblasts from normal and Alzheimer's donors.

Previous studies have reported that resting concentrations of intracellular calcium ion were markedly reduced in cultured dermal fibroblasts from Alzheimer's disease patients and that the ability of these cells to respond to serum stimulation was also decreased as compared to both young and age-matched control cells. In this study we have carefully reexamined these parameters in several of the same lines of fibroblasts and fail to find major differences in resting cytosolic calcium [Ca2+]i, in the response of [Ca2+]i to serum stimulation or in cell spreading in the AD cells as compared to young controls. The present findings suggest that cytoplasmic ionic calcium levels are neither pathognomonic for Alzheimer's cells nor of diagnostic value.     

ATP increases [Ca2+]i and ion secretion via a basolateral P2Y-receptor in rat distal colonic mucosa

 Under resting conditions the mammalian distal colon is a NaCl-absorptive epithelium. NaCl absorption occurs at surface cells in colonic crypts. Intracellular Ca²⁺ or cAMP are important second messengers that activate NaCl secretion, a function that is most pronounced in crypt bases. In the present study we examined the effect of extracellular ATP on isolated crypts of rat distal colon using the fura-2 technique. Intracellular Ca²⁺ ([Ca²⁺]_i) was measured spectrofluorimetrically either by photon counting or video imaging. ATP reversibly increased [Ca²⁺]_i in crypt base cells with an EC₅₀ of 4.5 μmol/l (n = 11). This [Ca²⁺]_i increase was composed of an initial peak, reflecting intracellular store release, and a secondary plateau phase reflecting transmembrane influx. Digital video imaging revealed that agonist-induced [Ca²⁺]_i elevations were most marked at the crypt base. In the middle part of the crypt ATP induced smaller increases of [Ca²⁺]_i (peak and plateau) as compared to basal cells and in surface cells this [Ca²⁺]_i transient was even further reduced. Attempts to identify the relevant P₂-receptor demonstrated the following rank order of potency: 2MeS-ATP > ADP ≥ ATP >> AMP > UTP > AMP-PCP > adenosine. In Ussing chamber experiments ATP (1 mmol/l) functioned as a secretagogue, increasing transepithelial voltage (V _te) and equivalent short-circuit current (I _sc): ΔI _sc = –36.4 ± 5.4 μA/cm², n = 17. Adenosine itself (1 mmol/l) induced an increase of I _sc of similar magnitude to that induced by ATP: ΔI _sc = –55.1 ± 8.4 μA/cm², n = 9. The effect of adenosine, but not that of ATP, was fully inhibited by the A₁/A₂-receptor antagonist 8-(p-sulphophenyl)theophylline, 0.5 mmol/l, n = 4. Together these data indicate that: (1) basolateral ATP induces [Ca²⁺]_i in isolated rat colonic crypts and acts as a secretagogue in the distal rat colon; (2) a basolateral P2Y-receptor is responsible for this ATP-induced NaCl secretion; (3) the ability of ATP to increase I _sc in Ussing chamber experiments is not mediated via adenosine; and (4) the agonist-induced [Ca²⁺]_i signals are mostly located in the crypt base, which is the secretory part of the colonic crypt. Received: 17 September 1996 / Received after revision: 20 January 1997 / Accepted: 28 January 1997     

Fluorescence and Laser Photon Counting: Measurements of Epithelial [Ca2+]i or [Na+]i with Ciliary Beat Frequency

We describe a system we developed that enabled simultaneous measurements of either epithelial calcium ion concentration ([Ca²⁺]i) or sodium ion concentration [Na⁺]i with the ciliary beat frequency (CBF) in native ciliated epithelia using either Fura-2 (AM) or SBFI (AM) ratiometric fluorescence photon counting along with nonstationary laser light scattering. Studies were performed using native epithelial tissues obtained from ovine tracheae. The dynamic range of the laser light-scattering system was determined by a simulated light beating experiment. The nonstationary CBF was demonstrated by the time-frequency analysis of the raw photon count sequences of backscattered heterodyne photons from cultured and native epithelia. Calibrations of calcium and sodium ion concentrations were performed using the respective Fura-2 and SBFI impermanent salts as well as in native epithelia. The cumulative responses of 10^–6, 10^–5, and 10^–4} M nifedipine on [Ca²⁺]i together with the CBF as well as the cumulative responses of 10^–5, 10^–4, and 10^–3 M amiloride on [Na⁺]i together with the CBF were also determined. Nifedipine decreased [Ca²⁺]i but had no effect on CBF. Amiloride decreased [Na⁺]i and CBF. Stimulation of CBF corresponded with either an increase of [Na⁺]i or an increase of [Ca²⁺]i. Decreases of [Na⁺]i or substantial decreases of [Ca²⁺]i were associated with decreases in the CBF. These data demonstrate the utility of this system for investigating the regulatory mechanisms of intracellular ions dynamics and the CBF in native epithelia. © 1998 Biomedical Engineering Society. PAC98: 8780+s, 8722-q, 4262Be     

An increase in [Ca2+]i activates basolateral chloride channels and inhibits apical sodium channels in frog skin epithelium

 The aim of this study was to investigate the mechanisms by which increases in free cytosolic calcium ([Ca²⁺]_i) cause a decrease in macroscopic sodium absorption across principal cells of the frog skin epithelium. [Ca²⁺]_i was measured with fura-2 in an epifluorescence microscope set-up, sodium absorption was measured by the voltage-clamp technique and cellular potential was measured using microelectrodes. The endoplasmic reticulum calcium-ATPase inhibitor thapsigargin (0.4 μM) increased [Ca²⁺]_i from 66 ± 9 to 137 ± 19 nM (n = 13, P = 0.002). Thapsigargin caused the amiloride-sensitive short circuit current (I _sc) to drop from 26.4 to 10.6 μA cm^–2 (n = 19, P<0.001) concomitant with a depolarization of the cells from –79 ± 1 to –31 ± 2 mV (n = 18, P<0.001). Apical sodium permeability (P ^a _Na) was estimated from the current/voltage (I/V) relationship between amiloride-sensitive current and the potential across the apical membrane. P ^a _Na decreased from 8.01·10^–7 to 3.74·10^–7 cm s^–1 (n = 7, P = 0.04) following an increase in [Ca²⁺]_i. A decrease in apical sodium permeability per se would tend to decrease I _sc and result in a hyperpolarization of the cell potential and not, as observed, a depolarization. Serosal addition of the chloride channel inhibitors 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS), diphenylamine-2-carboxylate (DPC), indanyloxyacetic acid 94 (IAA-94) and furosemide reversed the depolarization induced by thapsigargin, indicating that chloride channels were activated by the increase in [Ca²⁺]_i. This was confirmed in wash-out experiments with ³⁶Cl where it was shown that thapsigargin increased the efflux of chloride from 32.49 ± 5.01 to 62.63 ± 13.3 nmol·min^–1 cm^–2 (n = 5, P = 0.04). We conclude that a small increase in [Ca²⁺]_i activates a chloride permeability and inhibits the apical sodium permeability. The activation of chloride channels and the closure of apical sodium channels will tend to lower the macroscopic sodium absorption. Received: 25 June 1996 / Received after revision: 28 August 1996 / Accepted: 2 September 1996     

口服左旋精氨酸对血小板胞浆内钙离子浓度的影响

为观察口服左旋精氨酸对正常人血小板胞浆内钙离子浓度的影响并探讨其作用机制,选择了10名至少2周内未服用任何药物,不吸烟、喝酒的健康男性志愿者.受试者服药前做1次检测,然后口服左旋精氨酸4天,前3天每天服20克,分3次服用,第4天服用1次,服7克,3小时后抽血测试.结果表明,服用左旋精氨酸后,血小板胞浆内cGMP浓度(1.91±0.20pmol/109血小板vs 2.14±0.34pmol/109血小板,P＜0.05)、血清一氧化氮代谢产物NO-3浓度(32.66±14.31μmol/L vs 49.02±9.75μmol/L,P＜0.05)升高.ADP诱导的血小板[Ca2+]i升高幅度(A/B值0.45±0.14 vs 0.32±0.09, P＜0.01)降低.研究结果表明,口服左旋精氨酸可以通过促进血管内皮细胞和血小板合成NO来降低血小板胞浆内钙离子浓度的升高,从而抑制正常人血小板的活化.     

缺氧性神经干细胞钙离子浓度变化和葡萄糖保护作用的研究

为了从钙离子角度探讨葡萄糖对神经干细胞缺氧性损伤保护作用机制,本实验将三气培养箱的氧气浓度调至5%制备缺氧环境。分别在缺氧前后于无血清培养基中加入不同剂量的葡萄糖,同时设常氧常糖正常对照。通过MTT法检测干细胞的存活和增殖情况,用激光扫描共聚焦显微镜和Fluo-3荧光探针标记技术检测神经干细胞内钙离子浓度。结果显示,缺氧前加入30mmol/L葡萄糖,神经干细胞的存活率和增殖率较常糖缺氧组明显增高,其胞内钙离子浓度显著低于常糖缺氧组。缺氧后再加入葡萄糖时,其保护作用不明显。本实验提示:缺氧前给予足够浓度的葡萄糖可通过抑制胞内钙超载机制对神经干细胞损伤起到一定的保护作用。     

Long-term effect of early discharge on sEPSC and [Ca2+]i in developing neurons

To study the long-term changes induced in immature rat cortical neuronal cultures by transient exposure to an Mg(2+)-free treatment, at cultured day 6, cells were assigned into three groups, based on the mediums they were transiently exposed to as follows: control group 1 (CONT1) was exposed to Dulbecco's Modified Eagle's Medium (DMEM), control group 2 was exposed to a physiological solution (PS), and the magnesium-free physiological solution group (MGFPS) was exposed to the same medium as CONT2 except for the removal of magnesium. Following a 3-h exposure, the amplitude and frequency of spontaneous excitatory postsynaptic currents (sEPSC) were recorded and intracellular calcium concentrations ([Ca2+]i) were measured. Compared to the CONT1 and CONT2 groups, the MGFPS group displayed a significantly greater amplitude (at d6, d7, d9, and d12) and frequency (at d6, d7, and d9) of sEPSC (p<0.05). Also, both the resting and peak intracellular calcium levels were significantly greater in the MGFPS group at days 6, 7, 9, 12 and 17 (p<0.05). The rise time (time from resting level to peak level of intracellular calcium following NMDA application) was significantly shorter in the MGFPS group at culture days 7 and 17 and significantly longer at culture day 12 (p<0.05). Finally, we compared the percentage of cortical neurons expressing neuron-specific enolase (NSE) and found that there were no significant differences in the number of NSE positive neurons among three groups at days 7, 12, and 17. Our results suggests that there are long-term changes in sEPSCs and [Ca2+]i in cultured rat cortical neurons following exposure to Mg2+-free environment without cell loss.     

ATP induces transient elevations of [Ca2+]i in human neutrophils and primes these cells for enhanced O2- generation

ATP, when added to human polymorphonuclear neutrophils (PMNs) at concentrations similar to those attained extracellularly at sites of platelet thrombus formation (0.1 to 20 microM), causes an enhancement of N-formyl(methionyl)leucylphenylalanine (FMLP)-stimulated superoxide anion (O2-) generation. However, ATP by itself is an ineffective agonist for O2- generation by PMNs. The ATP-induced enhancement of O2- generation is associated with a shortened lag time in the response of PMNs to FMLP without a change in the median effective dose for FMLP, suggesting that signal transduction, rather than altered receptor affinity, is responsible for the enhanced oxidative response. Maximum enhancement of O2- generation is detected as early as 15 seconds and is maintained for at least 10 minutes. Of various nucleotides and nonhydrolyzable-ATP analogs test d, only ATP, UTP, and ITP were found to cause enhanced O2- generation by PMNs. Addition of ATP to quin2-loaded PMNs, in the absence of other stimuli, elicits a dramatic rise in [Ca2+]i which reaches a maximum of 500 to 800 nM at 30 seconds and slowly returns to baseline over 5 minutes. This ATP-induced rise in intracellular free Ca2+ concentration is correlated with the enhancement of FMLP-stimulated O2- generation both with respect to dose and nucleotide specificity. Stimulated Ca2+ uptake, rather than mobilization of intracellular Ca2+ stores, appears to be primarily responsible for the rise in intracellular free Ca2+ concentration. These studies indicate that an ATP-induced rise in intracellular free Ca2+ concentration, although insufficient by itself to elicit O2- generation by PMNs, is associated with a priming of PMNs for enhanced O2- generation when stimulated by other agonists.     

红车轴草提取物对小鼠淋巴细胞[Ca2+]i及腹腔巨噬细胞NO分泌和吞噬的影响

目的探讨红车轴草提取物(Trifoliumpratense Leguminosae extract,TLE)体外对小鼠淋巴细胞[Ca2+]i及腹腔巨噬细胞NO分泌和吞噬微球的影响。方法无菌条件下制备小鼠淋巴细胞悬液及小鼠腹腔巨噬细胞悬液;MTT法检测药物对细胞悬液的毒性情况;Fluo-4/AM染色结合流式细胞术分析TLE对小鼠淋巴细胞[Ca2+]i的影响;Griess反应系统检测TLE对脂多糖(LPS)刺激的小鼠腹腔巨噬细胞NO分泌的影响;1μm与2μm直径的荧光微球结合流式细胞术分析TLE对小鼠腹腔巨噬细胞吞噬作用的影响。结果终质量浓度为20、40mg/L的TLE对细胞的毒性小;TLE促进了淋巴细胞的Ca2+内流;TLE抑制了巨噬细胞的NO分泌与吞噬作用,与非TLE组比较P<0.01。结论对淋巴细胞[Ca2+]i及巨噬细胞的NO分泌和吞噬的作用可能是TLE调节小鼠免疫系统的途径。     

内皮素对成年鼠离体心室肌细胞内游离钙浓度的影响及机制

目的和方法：采用分离的Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙ大鼠心室肌细胞,以Ｆｕｒａ－２／ＡＭ荧光指示剂负载,检测心肌细胞内游离钙浓度（［Ｃａ２＋］ｉ）变化,探讨内皮素－１（ＥＴ－１）对［Ｃａ２＋］ｉ的作用及其机制。结果：ＥＴ－１（１×１０－７ｍｏｌ／Ｌ）引起［Ｃａ２＋］ｉ升高分两个时相：快速相和持续相,可被ＥＴＡ的特异性受体阻断剂ＢＱ１２３（２×１０－６ｍｏｌ／Ｌ）所阻断。移去细胞外液钙以及用百日咳毒素（２００ｎｇ／ｍＬ）处理１０ｈ后,ＥＴ－１仍引起快速相,但持续相消失。Ｒｙａｎｏｄｉｎｅ（４μｍｏｌ／Ｌ）和异搏定（２×１０－５ｍｏｌ／Ｌ）对ＥＴ－１诱导［Ｃａ２＋］ｉ升高的作用无显著影响。结论：ＥＴ－１升高［Ｃａ２＋］ｉ是通过ＥＴＡ受体介导;快速相［Ｃａ２＋］ｉ升高主要由胞内Ｒｙａｎｏｄｉｎｅ不敏感的钙池释放造成,与百日咳毒素敏感的Ｇ蛋白无关;持续相［Ｃａ２＋］ｉ升高主要由胞外Ｃａ２＋内流引起,不是通过电压依赖性Ｌ－型钙通道介导,与百日咳毒素敏感的Ｇ蛋白有关     

银杏内酯B对体外培养的视网膜神经细胞内钙离子浓度和线粒体功能的影响

目的： 观察银杏内酯B对体外培养的大鼠视网膜神经细胞内钙离子浓度和线粒体功能的影响。方法: 采用体外原代培养的大鼠视网膜神经细胞,建立谷氨酸损伤的视网膜神经细胞凋亡模型,与银杏内酯B共同培养,用激光扫描共聚焦显微镜检测对视网膜神经细胞内钙离子浓度和线粒体膜电位的影响。结果: 谷氨酸（8 mmol/L）作用后,视网膜神经细胞存活率降低,细胞凋亡增加,细胞内钙离子浓度增加,线粒体膜电位下降。GB干预后,钙离子浓度降低,线粒体膜电位显著升高,细胞凋亡明显减少。结论: GB能对抗谷氨酸兴奋性毒性, 保护视网膜神经细胞,这一作用可能是通过降低细胞内钙离子浓度和升高线粒体膜电位来实现的。     

Selective induction of LTP and LTD by postsynaptic [Ca2+]i elevation

Long-term potentiation (LTP) and long-term depression (LTD), two prominent forms of synaptic plasticity at glutamatergic afferents to CA1 hippocampal pyramidal cells, are both triggered by the elevation of postsynaptic intracellular calcium concentration ([Ca2+]i). To understand how one signaling molecule can be responsible for triggering two opposing forms of synaptic modulation, different postsynaptic [Ca2+]i elevation patterns were generated by a new caged calcium compound nitrophenyl-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid in CA1 pyramidal cells. We found that specific patterns of [Ca2+]i elevation selectively activate LTP or LTD. In particular, only LTP was triggered by a brief increase of [Ca2+]i with relatively high magnitude, which mimics the [Ca2+]i rise during electrical stimulation typically used to induce LTP. In contrast, a prolonged modest rise of [Ca2+]i reliably induced LTD. An important implication of the results is that both the amplitude and the duration of an intracellular chemical signal can carry significant biological information.     

大麻素抑制背根节神经元ATP诱发的[Ca~(2+)]i升高

目的:观察大麻素对背根节神经元ATP诱发的[Ca~(2+)]i升高的影响及机制。方法:培养SD大鼠背根节神经元,采用激光共聚焦技术检测培养神经元[Ca~(2+)]i的变化。结果:ATP(100μmol/L)经P2X受体介导可导致培养的背根节神经元[Ca~(2+)]i增高(P0.05);大麻素受体激动剂CP55940预孵育10 min可剂量依赖性地抑制背根节神经元ATP所致的[Ca~(2+)]i升高(P0.05);CB1受体(cannabinoid receptor 1,B1R)的拮抗剂AM251(10μmol/L)、CB2受体(Cannabinoid receptor 2,CB2R)的拮抗剂AM630(10μmol/L)均可显著降低CP55940(1μmol/L)的抑制效应(P0.05);腺苷酸环化酶激动剂Forskolin(10μmol/L)可逆转CP55940对ATP的抑制作用(P0.05)。结论:CP55940可显著抑制背根节神经元ATP诱发的[Ca~(2+)]i升高,CP55940的抑制效应可能是由CB1、CB2受体介导抑制背根节神经元PKA活性所致。     

Mechanisms underlying changes of tetanic [Ca2+]i and force in skeletal muscle

Force development in skeletal muscle is driven by an increase in myoplasmic free [Ca²⁺] ([Ca²⁺]_i) due to Ca²⁺ release from the sarcoplasmic reticulum (SR). The magnitude of [Ca²⁺]_i elevation during stimulation depends on: (a) the rate of Ca²⁺ release from the SR, (b) the rate of Ca²⁺ uptake by the SR, and (c) the myoplasmic Ca²⁺ buffering. We have used fluorescent Ca²⁺ indicators to measure [Ca²⁺]_i in intact, single fibres from mouse and Xenopus muscles under conditions where one or more of the above factors are changed. The following interventions resulted in increased tetanic [Ca²⁺]_i: β-adrenergic stimulation, which potentiates the SR Ca²⁺ release; application of 2,5-di(tert-butyl)-1,4-benzohydroquinone, which inhibits SR Ca²⁺ pumps; application of caffeine, which facilitates SR Ca²⁺ release and inhibits SR Ca²⁺ uptake; early fatigue, where the rate of SR Ca²⁺ uptake is reduced; acidosis, which reduces both the myoplasmic Ca²⁺ buffering and the rate of SR Ca²⁺ uptake. Reduced tetanic [Ca²⁺]_i was observed in late fatigue, due to reduced SR Ca²⁺ release, and in alkalosis, due to increased myoplasmic Ca²⁺ buffering. Force is monotonically related to [Ca²⁺]_i, but depends also on the myofibrillar Ca²⁺ sensitivity and the maximum force cross-bridges can produce. This is clearly illustrated by changes of intracellular pH where, despite a lower tetanic [Ca²⁺]_i, tetanic force is higher in alkalosis than acidosis due to increases of myofibrillar Ca²⁺ sensitivity and maximum cross-bridge force.     

Role of [Ca2+]i in the ATP-induced heat sensitization process of rat nociceptive neurons.

In inflamed tissue, nociceptors show increased sensitivity to noxious heat, which may account for heat hyperalgesia. In unmyelinated nociceptive afferents in rat skin in vitro, a drop of heat threshold and an increase in heat responses were induced by experimental elevation of intracellular calcium ([Ca2+]i) levels with the calcium ionophore ionomycin (10 microM). Similar results were obtained in experiments employing [Ca2+]i release from preloaded "caged calcium" (NITR-5/AM) via UV photolysis. In both cases, sensitization was prevented by preventing rises in [Ca2+]i with the membrane-permeant calcium chelator BAPTA-AM (1 mM). No pronounced change of mechanical sensitivity was observed. Heat-induced membrane currents (Iheat) were investigated with patch-clamp recordings, and simultaneous calcium measurements were performed in small sensory neurons isolated from adult rat dorsal root ganglia (DRG). Ionomycin-induced rises in [Ca2+]i resulted in reversible sensitization of Iheat. In the same subset of DRG neurons, the endogenous algogen ATP (100 microM) was used to elevate [Ca2+]i, which again resulted in significant sensitization of Iheat. In correlative recordings from the skin-nerve preparation, ATP induced heat sensitization of nociceptors, which again could be blocked by preincubation with BAPTA-AM. Rises in [Ca2+]i in response to inflammatory mediators, e.g., ATP, thus appear to play a central role in plastic changes of nociceptors, which may account for hypersensitivity of inflamed tissue.     

Effect of intracellular pH on agonist-induced [Ca2+]i transients in HT29 cells

 In this study we examined the influence of intracellular pH (pH_i) on agonist-induced changes of intracellular Ca²⁺ activity ([Ca²⁺]_i) in HT₂₉ cells. pH_i and [Ca²⁺]_i were measured microspectrofluorimetrically using BCECF and fura-2, respectively. Buffers containing trimethylamine (TriMA), NH₃/NH₄ ⁺ and acetate were used to clamp pH_i to defined values. The magnitudes of the peak and plateau of [Ca²⁺]_i transients induced by carbachol (CCH, 10^–6 mol/l) were greatly enhanced by an acidic pH_i and nearly abolished by an alkaline pH_i. The relationship between pH_i and the [Ca²⁺]_i peak was nearly linear from pH_i 7.0 to 7.8. This effect of pH_i was also observed at higher CCH concentrations (10^–4 and 10^–5 mol/l), at which the inhibitory effect of an alkaline pH_i was more pronounced than the stimulatory effect of an acidic pH_i. An acidic pH_i shifted the CCH concentration/response curve to the left, whereas an alkaline pH_i led to a rightward shift. The influence of pH_i on [Ca²⁺]_i transients induced by neurotensin (10^–8 mol/l) or ATP (5 × 10^–7 mol/l) was similar to its influence on those induced by CCH, but generally not as pronounced. Measurements of cellular inositol 1,4,5-trisphosphate (InsP ₃) showed no changes in response to acidification with acetate (20 mmol/l) or alkalinization with TriMA (20 mmol/l). The InsP ₃ increase induced by CCH was unaltered at an acidic pH_i, but was augmented at an alkaline pH_i. Confocal measurements of cell volume showed no significant changes induced by TriMA or acetate. Slow-whole-cell patch-clamp experiments showed no additional effect of CCH on the membrane voltage (V _m) measured after TriMA or acetate application. We conclude that pH_i is a physiological modulator of hormonal effects in HT₂₉ cells, as the [Ca²⁺]_i responses to agonists were significantly changed at already slightly altered pH_i. The measurements of InsP ₃, cell volume and V _m show that pH_i must act distally to the InsP ₃ production, and not via changes of cell volume or V _m. Received: 21 March 1997 / Received after revision: 14 May 1997 / Accepted: 15 May 1997     

汉防己碱与1, 6-二磷酸果糖对兴奋性氨基酸所致突触体内游离钙浓度升高的影响

目的:研究汉防己碱(Tet)与1, 6-二磷酸果糖(FDP)对兴奋性氨基酸(EAA)诱导的突触体内游离钙浓度升高的影响。方法:制备大鼠脑突触体, 以荧光法测定突触体内游离钙浓度;加入谷氨酸(Glu, 100μmol·L^-1), 天冬氨酸(Asp, 100μmol·L^-1), N-甲基-D-天冬氨酸(NMDA, 100μmol·L^-1)及Glu+Asp(50μmol·L^-1+50μmol·L^-1)后, 测定突触体内游离钙浓度的变化;提前给予Tet(10、30、60μmol·L^-1), FDP(15、30、75、150μmol·L^-1), MK-801(10、20μmol·L^-1)及Tet+FDP(10+15, 30+30μmol·L^-1)预孵, 再加入上述浓度的Glu、Asp、NMDA及Glu+Asp, 观察突触体内游离钙浓度的变化。结果:Glu、Asp、NMDA及Glu+Asp均呈剂量依赖性升高突触体内游离钙浓度, 提前给予不同浓度的Tet、FDP、MK-801及Tet+FDP, 均可使突触体内游离钙浓度呈剂量依赖性降低, 且以Tet+FDP的作用最明显。结论:Tet与FPD可显著抑制EAA诱导的突触体内游离钙浓度的升高, 这可能是二者保护缺血脑细胞的作用机制之一。Tet与FDP联合应用可能对缺血脑细胞有更强的保护作用。     

Partial uncoupling of neurotransmitter release from [Ca2+]i by membrane hyperpolarization

The dependence of evoked and asynchronous release on intracellular calcium ([Ca2+]i) and presynaptic membrane potential was examined in single-release boutons of the crayfish opener neuromuscular junction. When a single bouton was depolarized by a train of pulses, [Ca2+]i increased to different levels according to the frequency of stimulation. Concomitant measurements of evoked release and asynchronous release, from the same bouton, showed that both increased in a sigmoidal manner as a function of [Ca2+]i. When each of the depolarizing pulses was immediately followed by a hyperpolarizing pulse, [Ca2+]i was elevated to a lesser degree than in the control experiments, and the rate of asynchronous release and the quantal content were reduced; most importantly, evoked quantal release terminated sooner. The diminution of neurotransmitter release by the hyperpolarizing postpulse (HPP) could not be entirely accounted for by the reduction in [Ca2+]i. The experimental results are consistent with the hypothesis that the HPP reduces the sensitivity of the release machinery to [Ca2+]i, thereby not only reducing the quantal content but also terminating the quantal release process sooner.