PLASMA-PROTEINS MODIFY THE ENDOTHELIAL-CELL GLYCOCALYX OF FROG MESENTERIC MICROVESSELS
PLASMA-PROTEINS MODIFY THE ENDOTHELIAL-CELL GLYCOCALYX OF FROG MESENTERIC MICROVESSELS
复制标题
DOI：
10.1113/jphysiol.1992.sp018934
复制
发表时间：
1992-01-01
影响因子：
5.5
通讯作者：
CLOUGH, G
中科院分区：
文献类型：
--
作者：
ADAMSON, RH;CLOUGH, G
1. 我们研究了血浆蛋白与内皮细胞的相互作用，使用阳离子化铁蛋白作为细胞表面糖萼的标记物。在2.5%戊二醛中灌注固定之前，用玻璃微移液管依次灌注含有阳离子化铁蛋白（CF, 6.7 mg ml-1）的溶液（0.10 M-NaCl），然后用青蛙血浆或牛血清白蛋白（BSA， 50或10 mg ml-1）或无蛋白的林格氏溶液。一层CF，通常为2 - 4分子厚，与管腔内皮细胞表面相关。在用无蛋白林格氏液冲洗后的血管中，CF层紧密粘附在管腔内皮的所有区域，包括质膜、囊泡膈、包被凹坑和裂隙入口。然而，当固定期间存在等离子体时，CF层在所有区域与细胞表面分离达100 nm。在用BSA冲洗后的血管中，CF层也从膜上分离出来，但效果不太明显。利用灌注血管横切面（约50 nm厚）的电子显微图定量评估阳离子化铁蛋白与内皮细胞表面的关联，并以内皮细胞表面相关的CF层的深度、其与管腔内皮质膜的分离程度以及该层中CF的浓度来表达。血浆存在时的平均（+/- S.D.）分离度为32.3 +/- 10.5 nm (n = 12)，显著高于无蛋白林格液3.0 +/- 1.4 nm （n = 9）和牛血清白蛋白林格液8.3 +/- 3.0 nm （n = 8）。林格氏液中BSA的分离度也显著大于末灌注林格氏液（P < 0.01）。10和50 mg ml-1 BSA的效果没有差异。血浆存在时，糖萼总厚度（CF层厚度与分离层厚度之和）为56.2 +/- 13.7 nm，是Ringer溶液中28.0 +/- 9.1 nm的两倍（P < 0.01），而BSA的总厚度为30.9 +/- 5.4 nm，与Ringer溶液无显著差异。CF结合层内的铁蛋白浓度不受最终灌注溶液蛋白含量的显著影响，其范围在14.4 × 10(4)（林格溶液再灌注）和17.1 × 10(4) （50 mg ml-1 BSA再灌注）CF分子mu-m-3的平均值之间。内皮细胞表面CF层的深度也不受最终灌注液组成的影响，分别为24.8 +/- 8.8 nm、22.6 +/- 3.0 nm和23.9 +/- 7.0 nm，分别为林格液（n = 8）、林格液中BSA （n = 9）和血浆（n = 12）。我们得出结论，血浆蛋白的加入导致CF结合层从内皮细胞表面的质膜分离。我们对此的解释是，CF结合层的分离增加是CF结合的表面糖蛋白取向改变的结果。我们观察到整个血浆比单独使用血清白蛋白更有效，这与之前的观察结果一致，即血浆比单独使用其组成蛋白更有效地降低微血管通透性。
1. We have investigated the interaction of plasma proteins with the endothelial cell using cationized ferritin as a marker of the cell surface glycocalyx.2. Single microvessels of the frog mesentery were sequentially perfused using glass micropipettes with solutions containing cationized ferritin (CF, 6.7 mg ml-1) in 0.10 M-NaCl and then with either frog plasma or bovine serum albumin (BSA; 50 or 10 mg ml-1), or protein-free Ringer solution, before suffusion fixation in 2.5% glutaraldehyde.3. A layer of CF, usually two to four molecules thick, was associated with the luminal endothelial cell surface. In vessels post-flushed with protein-free Ringer solution the CF layer was closely adherent to all regions of the luminal endothelium, including the plasma membrane, vesicle diaphragms, coated pits and the entrances to clefts. However, when plasma was present during fixation the CF layer was separated from the cell surface by up to 100 nm over all regions. In vessels post-flushed with BSA the CF layer was also separated from the membrane but the effect was less striking.4. The association of cationized ferritin with the endothelial cell surface was assessed quantitatively using electron micrographs of transverse sections (approximately 50 nm thick) of the perfused vessels, and expressed in terms of the depth of the layer of CF associated with the endothelial cell surface, its separation from the plasma membrane of the luminal endothelium, and the concentration of CF in the layer. The mean (+/- S.D.) separation in the presence of plasma, 32.3 +/- 10.5 nm (n = 12), was significantly greater (P < 0.01) than that with either protein-free Ringer solution, 3.0 +/- 1.4 nm (n = 9), or BSA in Ringer solution, 8.3 +/- 3.0 nm (n = 8). The separation seen with BSA in Ringer was also significantly greater than that measured with a final Ringer solution perfusion (P < 0.01). The effects of 10 and 50 mg ml-1 BSA were not different from one another. The total glycocalyx thickness, defined as the sum of the separation layer and depth of CF layer, with plasma present, 56.2 +/- 13.7 nm, was twice the value seen with Ringer solution, 28.0 +/- 9.1 nm (P < 0.01), while the total thickness with BSA, 30.9 +/- 5.4 nm, was not different from the Ringer solution value.5. The concentration of ferritin within the CF binding layer was not significantly affected by the protein content of the final perfusate solution and ranged between mean values of 14.4 x 10(4) (Ringer solution reperfusion) and 17.1 x 10(4) (50 mg ml-1 BSA reperfusion) CF molecules mu-m-3. The depth of the CF layer at the endothelial cell surface also was unaffected by the composition of the final perfusate with values of 24.8 +/- 8.8 nm, 22.6 +/- 3.0 nm, and 23.9 +/- 7.0 nm for reperfusion with Ringer solution (n = 8), BSA in Ringer solution (n = 9) and plasma (n = 12), respectively.6. We conclude that addition of plasma proteins results in a separation of the CF binding layer from the plasma membrane of the endothelial cell surface. Our interpretation of this is that the increased separation of the CF binding layer is a result of a change in the orientation of surface glycoproteins to which CF binds. Our observation that whole plasma is more effective than serum albumin alone is consistent with previous observations that plasma is more effective at reducing microvascular permeability than its constituent proteins alone.