Francis S A, Kelly J M, McCormack J, Rogers R A, Lai J, Schneeberger E E, Lynch R D
Department of Pathology, Massachusetts General Hospital and Harvard Medical School, Boston 02114, USA.
Eur J Cell Biol. 1999 Jul;78(7):473-84. doi: 10.1016/s0171-9335(99)80074-0.
The role of plasma membrane lipids in regulating the passage of ions and other solutes through the paracellular pathway remains controversial. In this study we explore the contribution of cholesterol (CH) in maintaining the barrier function of an epithelial cell line using the CH-solubilizing agent methyl beta-cyclodextrin (MBCD) to stimulate CH efflux. Inclusion of 20 mM MBCD in both apical and basolateral media reduced CH levels by 70-80% with no significant effect on cell viability. Most of that decrease occurred during the first 30 min of incubation. Recovery of CH content to initial values was nearly complete 22 h after removal of MBCD. Within 30 min of adding MBCD to the culture medium, transepithelial electrical resistance (TER) increased, reaching maximum values 30-40% above controls. This early rise in TER occurred when MBCD was added to either side of the monolayer. The later rapid decline in TER was observed only when MBCD bathed the basolateral surface from which, coincidentally, CH efflux was most rapid. Freeze fracture replicas and transmission electron microscopy of monolayers exposed to MBCD for only 30 min revealed no increase in either the average tight junction (TJ) strand number or the dimensions of the lateral intercellular space. There was a statistically significant increase in the number of TJ particles associated with the E fracture face at this time. This raises the interesting possibility that during CH efflux there is a change in the interaction between TJ particles and underlying cytoskeletal elements. There was no change in staining for occludin and ZO-1. After exposing the basolateral surface to MBCD for 2 h, TER fell below control levels. The accompanying increase in mannitol flux suggests strongly that the decrease in TER resulted from an increase in the permeability of the paracellular and not the transcellular pathway. A decrease in immuno-staining for occludin and ZO-1 at TJs, a striking accumulation of actin at tri-cellular areas as well as a decline in the number of parallel strands, as seen in freeze fracture replicas, suggest that changes in cytoskeletal organization during long incubations with MBCD had physically disrupted the TJ network. Data are presented which suggest that the observed changes in paracellular permeability during CH efflux may be related to increased levels of lipid-derived second messengers, some of which may trigger changes in the phosphorylation status of TJ proteins.
质膜脂质在调节离子和其他溶质通过细胞旁途径的过程中所起的作用仍存在争议。在本研究中，我们使用胆固醇增溶剂甲基-β-环糊精（MBCD）来刺激胆固醇外流，探讨胆固醇（CH）在维持上皮细胞系屏障功能中的作用。在顶侧和基底外侧培养基中加入20 mM MBCD可使CH水平降低70 - 80%，而对细胞活力无显著影响。大部分降低发生在孵育的最初30分钟内。去除MBCD后22小时，CH含量几乎完全恢复到初始值。在向培养基中添加MBCD后30分钟内，跨上皮电阻（TER）增加，达到比对照高30 - 40%的最大值。当MBCD添加到单层的任一侧时，TER都会出现这种早期升高。仅当MBCD作用于基底外侧表面时，才观察到TER随后的快速下降，巧合的是，胆固醇从该表面外流最为迅速。对仅暴露于MBCD 30分钟的单层进行冷冻断裂复型和透射电子显微镜观察发现，平均紧密连接（TJ）链数量或细胞间侧向间隙的尺寸均未增加。此时，与E断裂面相关的TJ颗粒数量有统计学意义的增加。这就提出了一个有趣的可能性，即在胆固醇外流过程中，TJ颗粒与潜在的细胞骨架成分之间的相互作用发生了变化。闭合蛋白和ZO - 1的染色没有变化。在将基底外侧表面暴露于MBCD 2小时后，TER降至对照水平以下。伴随的甘露醇通量增加强烈表明，TER的降低是由于细胞旁途径而非跨细胞途径的通透性增加所致。如冷冻断裂复型所见，TJ处闭合蛋白和ZO - 1免疫染色减少、三细胞区域肌动蛋白显著积聚以及平行链数量减少，表明在与MBCD长时间孵育期间细胞骨架组织的变化已从物理上破坏了TJ网络。所呈现的数据表明，在胆固醇外流过程中观察到的细胞旁通透性变化可能与脂质衍生的第二信使水平增加有关，其中一些可能触发TJ蛋白磷酸化状态的变化。
