The cystic fibrosis transmembrane conductance regulator attenuates the endogenous Ca2+ activated Cl– conductance of Xenopus oocytes

 Oocytes from Xenopus laevis activate a Ca²⁺ dependent Cl^– conductance when exposed to the Ca²⁺ ionophore ionomycin. This Ca²⁺ activated Cl^– conductance (CaCC) is strongly outwardly rectifying and has a halide conductivity ratio (G_I– / G_Cl–) of about 4.4. This is in contrast to the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl^– conductance, which produces more linear I/V curves with a G_I– / G_Cl– ratio of about 0.52. Ionomycin enhanced CaCC (ΔG) in water injected and CFTR expressing ooyctes in the absence of 3-isobutyl-1-methylxanthine (IBMX, 1 mmol/l) by (μS) 23 ± 1.9 (n=9) and 23.6 ± 2.3 (n=11). Stimulation by IBMX did not change CaCC in water injected oocytes. CaCC was inhibited in CFTR-expressing ooyctes after stimulation with IBMX or a membrane permeable form of cAMP and was only 5.1 ± 0.48 μS (n=18) and 6.9 ± 0.6 (n=3), respectively. Inhibition of CaCC was correlated to the amount of CFTR-current activated by IBMX. ΔF508-CFTR which demonstrates only a small residual function in activating a cAMP dependent Cl^– channel in oocytes inhibited CaCC to a lesser degree (ΔG=12.1 ± 1.1 μS; n=7). Changes of CFTR and CaCC-Cl^– whole cell conductances were also measured when extracellular Cl^– was replaced by I^–. The results confirmed the reduced activation of CaCC in the presence of activated CFTR. No evidence was found for inhibition of CFTR-currents by increase of intracellular Ca²⁺. Moreover, intracellular cAMP was not changed by ionomycin and stimulation by IBMX did not change the ionomycin induced Ca²⁺ increase in Xenopus oocytes. Taken together, these results suggest that activation of CFTR-Cl^– currents is paralleled by an inhibition of Ca²⁺ activated Cl^– currents in ooyctes of Xenopus laevis. These results provide another example for CFTR-dependent regulation of membrane conductances other than cAMP-dependent Cl^– conductance. They might explain previous findings in epithelial tissues of CF-knockout mice. Received: 17 June 1997 / Received after revision: 4 September 1997 / Accepted: 5 September 1997     

Activation of ion transport by combined effects of ionomycin,forskolin and phorbol ester on cultured HT-29cl.19A human colonocytes

The differentiated clone 19A of the HT-29 human colon carcinoma cell line was used as a model to study the intracellular electrophysiological effects of interaction of the cAMP, the protein kinase C (PKC) and the Ca²⁺ pathways, (a) A synergistic effect between ionomycin and forskolin was observed. From intracellular responses it was concluded that the synergistic effect is caused by activation of an apical Cl^– conductance by protein kinase A and a basolateral K⁺ conductance by Ca²⁺. (b) A transient synergistic effect of ionomycin and the phorbol ester phorbol dibutyrate (PDB) was found. The decrease of the response appeared to be due to PKC-dependent inactivation of the basolateral K⁺ conductance. The synergism is caused by PKC-dependent increase of the apical Cl^– conductance and Ca²⁺-dependent increase of the basolateral K⁺ conductance. (c) The effects of carbachol and PDB were not fully additive presumably because of their convergence on PKC activation, (d) Forskolin and PDB, when added in this order, had a less than additive effect. Results of cell-attached patch-clamp studies, presented in the accompanying paper, showed a synergistic effect of forskolin and PDB on non-rectifying small-conductance Cl^– channels. Assuming that these channels are involved in the transepithelial responses it is suggested that forskolin and PDB induce a modulatory, synergistic increase of the apical Cl^– conductance when both pathways are activated simultaneously. (e) The HT-29cl.19A cells differ from T₈₄ cells in that the latter did not respond with an increase of the short-circuit current to addition of phorbol ester. This may be due to a very low expression of PKC.     

Vesicular demyelination induced by raised intracellular calcium

Incubation of nerve with high concentrations of the divalent cation ionophore A23187 produces myelin vesiculation (Schlaepfer 1977). This observation has now been extended using segments of rat ventral or dorsal root incubated with high (19 μM, 10 μg/ml) or low (1–1.5 μM) concentrations of A23187, or another divalent ionophore, ionomycin. Low concentrations of A23187 induced no vesiculation within a 2-h period. However, subsequent incubation of these roots in fresh, ionophore-free medium for 20 h, resulted in a prominent vesicular demyelination at the Schmidt-Lanterman incisures and paranodes of many fibres. At this time (22 h) the Schwann cells associated with some demyelinating internodes appeared vital upon ultrastructural examination: the cells also excluded the nuclear dye nigrosin. High concentrations of A23187 induced a similar vesicular demyelination in affected fibres within only 15–20 min. While the Schwann cells continued to exclude nigrosin for a further 4 h, their ultrastructural appearance indicated that they were probably in the early stages of necrosis. Incubation of moribund root with the ionophore produced no myelin vesiculation. At all ionophore concentrations, the myelin vesiculation was dependent upon the presence of extracellular Ca²⁺, and could be modulated in severity by varying this concentration. Other divalent cations (Ba²⁺, Co²⁺, Mg²⁺, Mn²⁺, Ni²⁺, Sr²⁺) could not substitute for Ca²⁺. The vesiculation induced by A23187 could be entirely prevented by the addition of Zn²⁺ (? 1 μM), Ni²⁺ (? 1–10 μM), Co²⁺ (? 100 μM) or Mn²⁺ (? 100 μM) to the bathing medium. A23187 applied to only part of an isolated internode resulted in a localization of the myelin disruption to that region. Ionomycin (? 1 μM), an ionophore with a greater selectivity for Ca²⁺ than A23187, also induced a prompt Ca²⁺-dependent myelin vesiculation. We conclude that vesicular demyelination can be initiated in vital Schwann cells by a raised intracellular Ca²⁺ concentration. Such demyelination does not necessarily lead to Schwann cell death. The possible relevance of the findings to vesicular demyelinating neuropathies is discussed, and a hypothesis regarding the mechanism of demyelination is advanced.     

The Physiology of the Transplanted Small Bowel: An Overview with Insight into Graft Function

Nilsson J, Sjödin L, Gylfe E. Supramaximal inhibition of cholecystokinin-induced pancreatic amylase release involves desensitization to cytoplasmic Ca²⁺. Scand J Gastroenterol 1994;29:561-568.

Background: Cholecystokinin (CCK) is a major stimulant of pancreatic enzyme secretion. The dose-response relationship for CCK-induced secretion is bell-shaped, with a characteristic supramaximal inhibition. The mechanism for this inhibition has now been studied.

Methods: The kinetics of amylase release and the changes of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]i) were recorded during stimulation of guinea-pig pancreatic acinar cells with different concentrations of cholecystokinin octapeptide (CCK-8) and the Ca²⁺ ionophore ionomycin. Results: Individual cells reacted with [Ca²⁺]_i oscillations at 10^?11 10^?10 M CCK-8 and with an initial peak followed by a sustained suprabasal level at 10 ^?9–10^?8 M of the agonist. The latter response was also seen in suspensions of acinar cells at all tested concentrations of CCK-8 and at 10^?6-10^?5 M of ionomycin. With increases of extracellular Ca²⁺ from 0.5 to 5.0 mM there was a rise of [Ca²⁺]_i during exposure to 10^?9-10^?8 M CCK-8 or 10^?5 M ionomycin but a paradoxical decrease at lower concentrations of CCK-8 or ionomycin. A dose-dependent increase of amylase release was seen at CCK-8 concentrations from 10^?11 to 10^?9M. At 10^?9-10^?8M CCK-8 secretion was characterized by an initial peak followed by a sustained phase. Whereas the initial peak of secretion remained unaffected by increasing CCK-8 from 10^?9 to 10^?8M, the sustained phase was inhibited (supramaximal inhibition). Increasing extracellular Ca²⁺ from 0.5 to 5.0 mM transiently enhanced secretion in response to 10^?9 M but lacked effect during supramaximal inhibition of secretion by 10^?8 M CCK-8.

Conclusions: Both initial and sustained CCK-8-stimulated amylase release increase with [Ca²⁺]_i. However, supramaximal inhibition of secretion was not due to a decrease of [Ca²⁺]_i but was characterized by desensitization to the stimulatory effect of [Ca²⁺]_i.     

离子霉素对SW480和SWO-38细胞中E-cadherin裂解的影响

 目的：观察可促进钙离子内流的工具药离子霉素(ionomycin),对不同肿瘤细胞株SW480及SWO-38其C末端片段2（E-cad/CTF2）的表达裂解的影响。方法：应用MTT法确定ionomycin作用于 SW480及SWO-38细胞的最佳浓度;Western blotting检测ionomycin 作用不同时间后E-cadherin全长及其C末端片段2(E-cad/CTF2)的表达水平;共聚焦显微镜动态检测SWO-38细胞胞内Ca2+浓度变化。结果：Ionomycin对SW480及SWO-38细胞均有细胞毒性作用,半数抑制浓度均为12 μmol/L,ionomycin可促进 SW480细胞中Ca2+浓度增加,E-cadherin裂解,E-Cad/CTF2片段水平升高,ionomycin没有引起SWO-38细胞中的大量钙内流,对E-cadherin裂解没有明显作用。结论：Ionomycin可促进钙离子内流,引起SW480肿瘤细胞E-cadherin裂解,但对SWO-38细胞E-cadherin裂解无明显影响。     

Calpeptin provides functional neuroprotection to rat retinal ganglion cells following Ca2+ influx

Apoptosis of retinal ganglion cells (RGCs) impairs vision in glaucoma patients. RGCs are also degenerated in multiple sclerosis (MS), resulting in loss of visual perception in MS patients. We examined the involvement of calpain and caspase cascades in apoptosis of the rat retinal ganglion cell line RGC-5 following 24 h of exposure to 250 nM ionomycin (IMN) or 300 units/ml interferon-gamma (IFN-gamma) and then evaluated functional neuroprotection with 2 microM calpeptin (CP, a calpain-specific inhibitor). Morphological and biochemical features of apoptosis were detected in RGC-5 cells following exposure to IMN or IFN-gamma. Fura-2 assay determined significant increases in intracellular free [Ca2+] following exposure to IMN or IFN-gamma. Pretreatment with CP for 1 h prevented Ca2+ influx, proteolytic activities, and apoptosis in RGC-5 cells. Western blot analyses showed an increase in activities of calpain and caspase-12, upregulation of Bax:Bcl-2 ratio, release of cytochrome c from mitochondria, and increase in caspase-9 and caspase-3 activities during apoptosis. Increased caspase-3 activity was also confirmed by a colorimetric assay. Activation of caspase-8 and cleavage of Bid to tBid in RGC-5 cells following exposure to IFN-gamma indicated co-operation between extrinsic and intrinsic pathways of apoptosis. Patch-clamp recordings showed that pretreatment with CP attenuated apoptosis and maintained normal whole-cell membrane potential, indicating functional neuroprotection. Taken together, our results demonstrated that Ca2+ overload could be responsible for activation of calpain and caspase cascades leading to apoptotic death of RGC-5 cells and CP provided functional neuroprotection.     

PHA与PMA对外周血单个核细胞产生IL-4、IFN——γ水平的研究

目的 :比较 PHA(植物血凝素 )与 PMA(佛波醇乙酯 )对 PBMCs(外周血单个核细胞 )的刺激效果。方法 :选择健康人群 ,取外周静脉血 ,分离得到 PBMCs,分别加入 PHA+ ionomycin(离子霉素 )及 PMA+ ionomycin进行刺激并孵育 2 h、4h、6h、12 h、2 4h、48h,采用双抗体夹心 ABC-ELISA法 ,分别测定不同时相培养上清中 IL -4和 IFN-γ水平。结果 :刺激 2 h后 ,PHA组与 PMA组 IFN-γ和 IL -4水平无差别 ;刺激 4h后 ,PMA组 IFN-γ和 IL-4水平均高于 PHA组 (P<0 .0 1) ;刺激 6h后 ,PHA组 IFN-γ水平低于 PMA组 (P<0 .0 5 ) ,IL -4水平高于 PMA组 (P<0 .0 5 ) ;刺激 12 h后 ,PHA组 IFN-γ与 PMA组接近 ,两组 IL -4水平亦无差别 ;刺激 2 4h和 48h后 ,PHA组 IFN-γ和 IL-4水平均高于 PMA组 (P<0 .0 5 )。结论 :PHA和 PMA对 PBMCs不同时间的刺激效果不同。     

流式细胞术检测大鼠Th1／Th2细胞的刺激方案研究

目的探讨流式细胞术检测大鼠Thl／Th2细胞时使用刺激剂的最佳方案。方法用不同浓度佛波酯（PMA）联合不同浓度离子霉素（Ion）作为激活剂，刺激大鼠外周血T淋巴细胞，采用流式细胞仪三色法方案。观察和分析在不同浓度组合的激活剂刺激下检测T淋巴细胞内干扰素-γ／（INF-γ）、白介素-4（IL-4）的分泌效果。结果PMA浓度为25ng／mL时，联合浓度分别为0、2、5、10、15、20μg／mL的Ion刺激大鼠淋巴细胞，胞内IFN-γ／IL-4的表达阳性率分别为（1．19±0．26）％／（2．46±0132）％、（1．23±0．21）％／（2．56±0．86）％、（3．08±0．56）％／（12．01±2．01）％、（4．01±O．48）％／（9．80±1．80）％、（3．98±1．78）％／（11．85±4．83）％、（3．76±1．08）％／（10．78±2．35）％。结论选用工作浓度为5～10μg／mL的Ion联合工作浓度为25ng／mL的PMA刺激培养大鼠淋巴细胞即可起到较好的激活作用，而且不会带来如CD4细胞下调、流式细胞术设门难度增加等干扰因素。在该浓度下，CD3／CD8和CD4两种设门方案均可用于大鼠Thl／Th2细胞检测。     

Sentinel node mapping identifies vaccine-draining lymph nodes with tumor-specific immunological activity

BACKGROUND: Adoptive immunotherapy (AIT) with 4T07-IL2 vaccine-draining lymph node (DLN) cells induced regression of established 4T07 mammary carcinomas, but contralateral non-DLN were inactive. These experiments were performed to determine whether mapping with isosulfan blue (IB), as described for identification of sentinel nodes, would identify vaccine-DLN with antitumor activity. METHODS: Ten days after vaccination with 4T07-IL-2, .1 ml of 1% IB was injected into the vaccination site (footpads or flanks). After 3 minutes, mice were euthanized, and the blue-stained nodes were collected. With flank vaccination, IB identified both an inguinal and an axillary node. We also collected DLNs blindly in mice not receiving IB dye. DLN cell suspensions were then activated with bryostatin 1, ionomycin, and IL-2, expanded in culture, and adoptively transferred to mice bearing established 4T07 flank tumors. RESULTS: Complete tumor regression occurred in nearly all mice treated with popliteal or inguinal DLNs collected with or without IB. IB-stained axillary DLNs cured 100% of tumor-bearing mice, whereas none of the mice treated with blindly collected axillary DLNs were cured. CONCLUSION: We have shown that IB identifies immunologically active DLNs, does not interfere with expansion of lymphocytes in vitro, and, more importantly, has no detrimental effect on the ability of lymphocytes to induce tumor regression in vivo. For axillary DLNs, use of IB mapping identified immunologically active lymph nodes that could not otherwise be found.     

佛波醇酯加离子霉素诱导脐血和成人外周血CD4+CD25+ T细胞分泌IL-2的相关机制研究

目的：以佛波醇酯加离子霉素作为刺激剂,验证CD4+CD25+ 调节性T细胞本身并不存在分泌IL-2障碍；同时通过对脐血和成人外周 血的比较性研究，了解脐血CD4+CD25+ T细胞的成熟度。方法：以au toMACS从足月婴儿脐血（CB）和成人外周血（PB）分选CD4+CD25+和CD4+CD25-T细 胞，以PDB+ionomycin作为刺激剂，培养45 h后流式细胞术检测各组细胞表达CD69和CD25水 平，并以Luminex多重细胞因子检测技术检测培养上清中7种细胞因子的浓度。结果:经PDB+ionomycin刺激后，CB、PB的CD4+CD25+ 和CD4+CD25- T细胞均 发生增殖，但在培养 45 h 后CD4+CD25+ T细胞均出现细胞状态变差或死亡倾向。C B、PB 的CD4+CD25+ T细胞活化后CD25分子表达进一步上调，高于CD25-细胞活化后的CD25分 子密度。经PDB+ionomycin刺激后，PB CD4+CD25+和CD4+CD25- T细胞均分泌高水平 的IFN-γ、IL-2和TNF-α，但CD25+ 细胞分泌IL-5、IL-4和IL-10水平远远高于CD25-细 胞；CB CD4+CD25+和CD4+CD25- T细胞亦分泌高水平的IL-2和TNF-α，但IFN-γ水 平远远低于PB，基本不分泌IL-5、IL-4和IL-10。结论：CD4+CD25+ T细胞本身并不存在合成和分泌IL-2障碍，其可能具有与传统T细胞不同的T细胞受体信息转 导模式；脐血CD4+CD25+ T细胞功能尚未完全成熟。