Stelzner T J, Weil J V, O'Brien R F
Cardiovascular Pulmonary Research Laboratory, University of Colorado Health Sciences Center, Denver 80262.
J Cell Physiol. 1989 Apr;139(1):157-66. doi: 10.1002/jcp.1041390122.
Cyclic adenosine monophosphate (AMP) has numerous important effects on cell structure and function, but its role in endothelial cells is unclear. Since cyclic AMP has been shown to affect transmembrane transport, cell growth and morphology, cellular adhesion, and cytoskeletal organization, it may be an important determinant of endothelial barrier properties. To test this we exposed bovine pulmonary artery endothelial cell monolayers to substances known to increase cyclic AMP and measured their effect on endothelial permeability to albumin and endothelial cell cyclic AMP concentrations. Cholera toxin (CT), a stimulant of the guanine nucleotide binding subunit of adenylate cyclase, led to a concentration-dependent 2-6-fold increase in cyclic AMP which was associated with a 3-10-fold reduction in albumin transfer across endothelial monolayers. The effect was not specific to albumin as similar barrier-enhancing effects were also noted with an unrelated macromolecule, fluorescein isothiocyanate (FITC)-dextran (MW 70,000). Barrier enhancement with cyclic AMP elevation was also observed with forskolin, a stimulant of the catalytic subunit of adenylate cyclase. The temporal pattern of barrier enhancement seen with these agents paralleled their effects on increasing cyclic AMP, and the barrier enhancement could be reproduced by incubation with either dibutyryl cyclic AMP or Sp-cAMPS, cyclic AMP-dependent protein kinase agonists. Furthermore, the forskolin effect on barrier enhancement was partially reversed with Rp-cAMPS, an antagonist of cyclic AMP-dependent protein kinase. Since endothelial actin polymerization may be an important determinant of endothelial barrier function, we sought to determine whether the cyclic AMP-induced effects were associated with increases in the polymerized actin pool (F-actin). Both cholera toxin and forskolin led to apparent endothelial cell spreading and quantitative increases in endothelial cell F-actin fluorescence. In conclusion, increased endothelial cell cyclic adenine nucleotide activity was an important determinant of endothelial barrier function in vitro. The barrier enhancement was associated with increased endothelial apposition and increases in F-actin, suggesting that influences on cytoskeletal assembly may be involved in this process.
环磷酸腺苷（AMP）对细胞结构和功能有许多重要影响，但其在内皮细胞中的作用尚不清楚。由于环磷酸腺苷已被证明会影响跨膜运输、细胞生长和形态、细胞黏附以及细胞骨架组织，它可能是内皮屏障特性的重要决定因素。为了验证这一点，我们将牛肺动脉内皮细胞单层暴露于已知能增加环磷酸腺苷的物质中，并测量它们对内皮细胞对白蛋白的通透性以及内皮细胞环磷酸腺苷浓度的影响。霍乱毒素（CT）是腺苷酸环化酶鸟嘌呤核苷酸结合亚基的刺激剂，可导致环磷酸腺苷浓度依赖性地增加2至6倍，这与白蛋白跨内皮单层转运减少3至10倍相关。这种效应并非白蛋白所特有，因为在一种不相关的大分子异硫氰酸荧光素（FITC）-葡聚糖（分子量70,000）中也观察到了类似的屏障增强效应。腺苷酸环化酶催化亚基的刺激剂福斯可林也观察到环磷酸腺苷升高导致屏障增强。这些药物引起的屏障增强的时间模式与其增加环磷酸腺苷的作用平行，并且通过与二丁酰环磷酸腺苷或Sp-cAMPS（环磷酸腺苷依赖性蛋白激酶激动剂）孵育可以重现屏障增强。此外，福斯可林对屏障增强的作用被环磷酸腺苷依赖性蛋白激酶拮抗剂Rp-cAMPS部分逆转。由于内皮肌动蛋白聚合可能是内皮屏障功能的重要决定因素，我们试图确定环磷酸腺苷诱导的效应是否与聚合肌动蛋白池（F-肌动蛋白）的增加有关。霍乱毒素和福斯可林都导致内皮细胞明显铺展以及内皮细胞F-肌动蛋白荧光定量增加。总之，体外内皮细胞环腺嘌呤核苷酸活性增加是内皮屏障功能的重要决定因素。屏障增强与内皮细胞贴附增加和F-肌动蛋白增加有关，表明对细胞骨架组装的影响可能参与了这一过程。
