Ghosh T K, Eis P S, Mullaney J M, Ebert C L, Gill D L
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore 21201.
J Biol Chem. 1988 Aug 15;263(23):11075-9.
The action of inositol 1,4,5-trisphosphate (InsP3) in releasing intracellular Ca2+ is shown to be competitively and potently antagonized by the glycosaminoglycan, heparin. Using either permeabilized cells of the DDT1MF-2 smooth muscle cell line, or an isolated microsomal membrane fraction derived from intact cells, heparin (4-6 kDa) at 10 micrograms/ml was observed to completely block the action of InsP3 in releasing Ca2+ accumulated via the ATP-dependent Ca2+ pump. In permeabilized cells, heparin had no effect on Ca2+ pump activity or on passive Ca2+ fluxes contributing to equilibrium Ca2+ accumulation. Heparin up to 100 micrograms/ml had no effect on the GTP-activated Ca2+ translocation process previously characterized in this cell line. Half-maximal inhibition of Ca2+ release activated by 10 microM InsP3 occurred with heparin at approximately 0.6 and 0.2 microgram/ml in permeabilized cells and isolated microsomes, respectively. Using microsomes, InsP3 dose-response curves in the presence and absence of 0.2 microgram/ml heparin (approximately 40 nM) revealed a 10-fold increase in apparent Km for InsP3 (0.31 microM in the absence of heparin) with no change in Vmax, indicating a competitive action of heparin. The results revealed a very high apparent affinity of heparin for the InsP3 active site, with a calculated Ki value of 2.7 nM. Heparin was shown to rapidly (within 20 s) reverse prior full activation of InsP3-mediated Ca2+ release returning the Ca2+ equilibrium back to that observed without InsP3. This reversal occurs even after prolonged (6 min) InsP3 activation. These results indicate a specific, high affinity, and competitive antagonism of the InsP3 active site by heparin. The rapidly induced reversal of InsP3-activated Ca2+ release by heparin strongly suggests that InsP3 directly activates a channel which remains open only while InsP3 is associated and closes immediately upon InsP3 dissociation.
已表明，糖胺聚糖肝素可竞争性且有效地拮抗肌醇1,4,5 -三磷酸（InsP3）释放细胞内Ca2+的作用。使用DDT1MF - 2平滑肌细胞系的透化细胞或源自完整细胞的分离微粒体膜组分，观察到10微克/毫升的肝素（4 - 6 kDa）可完全阻断InsP3释放通过ATP依赖性Ca2+泵积累的Ca2+的作用。在透化细胞中，肝素对Ca2+泵活性或对有助于平衡Ca2+积累的被动Ca2+通量没有影响。高达100微克/毫升的肝素对先前在该细胞系中表征的GTP激活的Ca2+转运过程没有影响。在透化细胞和分离的微粒体中，分别用约0.6微克/毫升和0.2微克/毫升的肝素对由10微摩尔/升InsP3激活的Ca2+释放产生半数最大抑制。使用微粒体，在存在和不存在0.2微克/毫升肝素（约40纳摩尔）的情况下，InsP3剂量 - 反应曲线显示InsP3的表观Km增加了10倍（在不存在肝素时为0.31微摩尔/升），而Vmax没有变化，表明肝素具有竞争性作用。结果显示肝素对InsP3活性位点具有非常高的表观亲和力，计算出的Ki值为2.7纳摩尔。已表明肝素可迅速（在20秒内）逆转InsP3介导的Ca2+释放的先前完全激活，使Ca2+平衡恢复到未用InsP3时观察到的状态。即使在InsP3长时间（6分钟）激活后这种逆转仍会发生。这些结果表明肝素对InsP3活性位点具有特异性、高亲和力和竞争性拮抗作用。肝素迅速诱导的InsP3激活的Ca2+释放的逆转强烈表明，InsP3直接激活一个通道，该通道仅在InsP3结合时保持开放，并在InsP3解离后立即关闭。
