Hemmerich S, Sijpkens D, Pecht I
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Biochemistry. 1991 Feb 12;30(6):1523-32. doi: 10.1021/bi00220a012.
Type I Fc epsilon receptor (Fc epsilon RI) mediated Ca2+ uptake and secretion of rat serosal mast cells have been shown to be inhibited by disodium 1,3-bis [(2'-carboxylatochromon-5'-yl) oxy]-2-hydroxypropane (disodium cromoglycate, DSCG), which is widely employed in the treatment of allergic asthma [Foreman et al. (1977) Br. J. Pharmacol. 59, 473P-474P; Cox (1967) Nature (London) 216, 1328-1329]. This drug was also found to modify the protein phosphorylation pattern of these mast cells. [Theoharides et al. (1980) Science 207, 80-82]. We have isolated by affinity chromatography on a water-insoluble cromoglycate-carrying matrix a cytosolic enzyme recently identified as a nucleoside 5'-diphosphate kinase. In order to examine a possible intracellular activity of the drug, a cell-permeant cromoglycate derivative, 1,3-bis [[2'-[[(acetoxymethyl)oxy]carbonyl]chromon-5'- yl]oxy]-2-hydroxypropane [bis(acetoxymethyl) cromoglycate, CG/AM], has been synthesized, and its uptake and effect on the Fc epsilon RI-mediated exocytosis of mast cells was investigated. A tritium-labeled CG/AM derivative, used as radioactive tracer, was found to permeate mucosal mast cells of the rat line RBL-2H3 and accumulate intracellularly up to 40-fold its extracellular concentration following hydrolysis by cytoplasmic hydrolases. A CG/AM dose dependent inhibition of the Fc epsilon RI-induced mediator secretion was observed in RBL-2H3 cells loaded with this compound (I50 approximately 40 microM extracellular CG/AM). A similar dose-dependent inhibition was observed for both the Fc epsilon RI-mediated transient rise in the concentration of cytosolic free Ca2+ ions [( Ca2+]i) and the net Ca2+ influx, as monitored by the fluorescent indicator Quin2 and the radioactive tracer 45Ca2+, respectively. These results clearly show that cell-permeant cromoglycate inhibits the Fc epsilon RI-mediated Ca2+ influx into the cell and further underscore the dominant role of this process in the coupling of stimulus to secretion in RBL cells. Furthermore, with the identification of nucleoside 5'-diphosphate kinase as a potential intracellular target for CG activity, distinct mechanisms of action may be inferred for cell-permeant and nonpermeant forms of CG.
I型Fcε受体（FcεRI）介导的大鼠浆膜肥大细胞的Ca2+摄取和分泌已被证明会受到1,3-双[（2'-羧基色酮-5'-基）氧基]-2-羟基丙烷二钠（色甘酸二钠，DSCG）的抑制，DSCG被广泛用于治疗过敏性哮喘[福尔曼等人（1977年）《英国药理学期刊》59卷，473P - 474P；考克斯（1967年）《自然》（伦敦）216卷，1328 - 1329页]。还发现这种药物会改变这些肥大细胞的蛋白质磷酸化模式。[西奥哈里德斯等人（1980年）《科学》207卷，80 - 82页]。我们通过在水不溶性携带色甘酸的基质上进行亲和层析，分离出一种最近被鉴定为核苷5'-二磷酸激酶的胞质酶。为了研究该药物可能的细胞内活性，合成了一种细胞可渗透的色甘酸衍生物，1,3-双[[2'-[[（乙酰氧基甲基）氧基]羰基]色酮-5'-基]氧基]-2-羟基丙烷[双（乙酰氧基甲基）色甘酸，CG/AM]，并研究了其对肥大细胞FcεRI介导的胞吐作用的摄取和影响。一种用作放射性示踪剂的氚标记CG/AM衍生物被发现可渗透大鼠RBL-2H3系的黏膜肥大细胞，并在被细胞质水解酶水解后在细胞内积累，其细胞内浓度高达细胞外浓度的四十倍。在加载了这种化合物的RBL-2H3细胞中观察到CG/AM对FcεRI诱导的介质分泌有剂量依赖性抑制作用（细胞外CG/AM的I50约为40微摩尔）。分别通过荧光指示剂喹啉2和放射性示踪剂45Ca2+监测发现，对于FcεRI介导的胞质游离Ca2+离子（[Ca2+]i）浓度的瞬时升高和净Ca2+内流，也观察到了类似的剂量依赖性抑制作用。这些结果清楚地表明，细胞可渗透的色甘酸抑制FcεRI介导的Ca2+流入细胞，并进一步强调了这一过程在RBL细胞中刺激与分泌偶联中的主导作用。此外，随着核苷5'-二磷酸激酶被鉴定为CG活性的潜在细胞内靶点，可以推断出细胞可渗透和不可渗透形式的CG有不同的作用机制。
