Meldolesi J, Castiglioni G, Parma R, Nassivera N, De Camilli P
J Cell Biol. 1978 Oct;79(1):156-72. doi: 10.1083/jcb.79.1.156.
Incubation of guinea pig pancreatic lobules in Ca++-free Krebs-Ringer bicarbonate solution (KRB) containing 0.5 mM ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) results in the progressive fragmentation of the occluding zonulae (ZO) with formation of multiple discrete junctions (fasciae occludentes) localized in the lateral and lumenal plasmalemma. After 1--2 h of such incubation, most ZO appear completely disassembled. This results in the disappearance of the heterogeneity in density of intramembrane particles on the P-fracture faces of the basolateral and lumenal plasmalemma. If Ca++ ions are reintroduced into the incubation fluid at this point, continous zonulae reform around the apices of the cells; in contrast, the density of intramembrane particles (imp) at the lumenal plasmalemma remains the same as in the basolateral region, at least for 3 h after Ca++ reintroduction. When added to the incubation fluid, cycloheximide (at a dose known to inhibit protein synthesis greater than 95%) and cytochalasin B (at doses which disrupt microfilaments and modify the cell shape) had no effect on the organization of ZO, on their disassembly in Ca++-free, EGTA medium, or on their Ca++-dependent reformation. Likewise, the organization and disassembly of ZO were unaffected by colchicine; however, after treatment with the latter drug the reassembly was defective, with formation of strand networks on the lateral surface and incomplete segregation of the lumenal region. Antimycin A, on the other hand, when added to the Ca++-EGTA medium, induced a large proliferation of long, infrequently anastomosed junctional strands, usually arranged to form ribbons, festoons, and other bizarre arrays. The possible relationship of these in vitro findings to the in vivo biogenesis and turnover of occluding junctions is discussed. It is suggested that the impairment of reassembly of zonulae by colchicine might be correlated with the disorder induced by the drug on the general organization of pancreatic exocrine cells. Moreover, antimycin A could act by promoting the aggregation of a pool of free junctional strand components (or precursors) that might exist normally in pancreatic exocrine cells.
将豚鼠胰腺小叶置于含0.5 mM乙二醇双（β-氨基乙醚）N，N，N'，N'-四乙酸（EGTA）的无钙Krebs-Ringer碳酸氢盐溶液（KRB）中孵育，会导致封闭小带（ZO）逐渐断裂，形成多个位于外侧和腔面膜质膜上的离散连接（紧密连接）。经过1至2小时的这种孵育后，大多数ZO似乎完全解体。这导致基底外侧和腔面膜质膜的P-断裂面上膜内颗粒密度的异质性消失。如果此时将Ca++离子重新引入孵育液中，连续的小带会在细胞顶端周围重新形成；相反，腔面膜质膜上的膜内颗粒（imp）密度至少在重新引入Ca++后的3小时内与基底外侧区域保持相同。当加入孵育液中时，环己酰亚胺（已知剂量可抑制蛋白质合成超过95%）和细胞松弛素B（剂量可破坏微丝并改变细胞形状）对ZO的组织、在无Ca++、EGTA培养基中的解体或其Ca++依赖性重新形成均无影响。同样，秋水仙碱对ZO的组织和解体也没有影响；然而，用后一种药物处理后，重新组装存在缺陷，外侧表面形成链状网络，腔区域的分离不完全。另一方面，抗霉素A加入到Ca++-EGTA培养基中时，会诱导大量长的、很少吻合的连接链增殖，通常排列成带状、花彩状和其他奇异的阵列。讨论了这些体外研究结果与体内紧密连接的生物发生和更新之间的可能关系。有人认为秋水仙碱对小带重新组装的损害可能与该药物对胰腺外分泌细胞总体组织的紊乱有关。此外，抗霉素A可能通过促进胰腺外分泌细胞中可能正常存在的一组游离连接链成分（或前体）的聚集而起作用。
