Unno N, Menconi M J, Fink M P
Department of Surgery, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
Surgery. 1997 Aug;122(2):485-91; discussion 491-2. doi: 10.1016/s0039-6060(97)90042-8.
Nitric oxide (NO.) increases the permeability of cultured intestinal epithelial monolayers. NO. reacts with superoxide anion to form peroxynitrite anion, which can be protonated under mildly acidic conditions to form the potent and versatile oxidizing agent, peroxynitrous acid. We hypothesized that intracellular acidosis induced by the Na(+)-H+ antiport blocker, amiloride, would favor the formation of peroxynitrous acid and thereby augment hyperpermeability induced by the NO. donor, SIN-1.
Caco-2BBe human intestinal epithelial monolayers were grown on permeable supports in bicameral chambers. The permeability of monolayers was assessed by measuring the transepithelial flux of fluorescein disulfonic acid (FS).
Incubation of monolayers with SIN-1 increased permeability to FS. Adding amiloride augmented SIN-1-induced hyperpermeability. SIN-1 plus amiloride also decreased cellular adenosine triphosphate content and caused derangements of the actin-based cytoskeleton as demonstrated by fluorescence microscopy. Coincubation of monolayers with several free-radical or peroxynitrous acid scavengers (deferoxamine, mannitol, dimethyl sulfoxide, or ascorbate) ameliorated hyperpermeability induced by SIN-1 plus amiloride.
Amiloride augments NO.-induced intestinal epithelial permeability, apparently by promoting the development of intracellular acidosis and thereby favoring the formation of the peroxynitrous acid.
一氧化氮（NO.）可增加培养的肠上皮单层的通透性。NO.与超氧阴离子反应形成过氧亚硝酸盐阴离子，在轻度酸性条件下可质子化形成强效且多功能的氧化剂过氧亚硝酸。我们推测，钠氢反向转运体阻滞剂阿米洛利诱导的细胞内酸中毒会促进过氧亚硝酸的形成，从而增强由NO.供体SIN-1诱导的高通透性。
将Caco-2BBe人肠上皮单层培养在双室腔室的可渗透支持物上。通过测量荧光素二磺酸（FS）的跨上皮通量来评估单层的通透性。
用SIN-1孵育单层会增加对FS的通透性。添加阿米洛利会增强SIN-1诱导的高通透性。SIN-1加阿米洛利还会降低细胞三磷酸腺苷含量，并导致基于肌动蛋白的细胞骨架紊乱，荧光显微镜检查证实了这一点。将单层与几种自由基或过氧亚硝酸清除剂（去铁胺、甘露醇、二甲基亚砜或抗坏血酸）共同孵育可改善由SIN-1加阿米洛利诱导的高通透性。
阿米洛利增强NO.诱导的肠上皮通透性，显然是通过促进细胞内酸中毒的发展，从而有利于过氧亚硝酸的形成。
