Osypiw J C, Gleeson D, Lobley R W, Pemberton P W, McMahon R F
Department of Gastroenterology, Royal Infirmary, Manchester.
Exp Physiol. 1994 Sep;79(5):723-39. doi: 10.1113/expphysiol.1994.sp003803.
Acid-base transport systems have been incompletely characterized in intact intestinal epithelial cells. We therefore studied the human cell line Caco-2, cultured on Teflon membranes to form confluent monolayers with apical microvilli on transmission electron microscopy and progressive enrichment in microvillar hydrolases. Monolayers (16- to 25-day-old), loaded with the pH-sensitive dye BCECF-AM (2',7'-bis (carboxyethyl)-5-carboxyfluorescein), were mounted in a spectrofluorometer cuvette to allow selective superfusion of apical and basolateral surfaces with Hepes- or HCO(3-)-buffered media. Intracellular pH (pHi) was measured by dual-excitation spectrofluorimetry; calibration was with standards containing nigericin and 110 mM K+ corresponding to measured intracellular [K+] in Caco-2 cell monolayers. In HCO(3-)-free (Hepes-buffered) media, bilateral superfusion with 1 mM amiloride or with Na(+)-free media reversibly inhibited pHi recovery from an intracellular acid load (NH4Cl pulse) by 86 and 98% respectively. Selective readdition of Na+ to the apical or basolateral superfusate also induced a pHi recovery, which was inhibited by ipsilateral but not by contralateral amiloride (1 mM). The pHi recovery induced by apical Na+ readdition had a Michaelis constant (Km) for Na+ of 30 mM and a relatively high inhibitor constant (Ki) for amiloride of 45.5 microM. Initial pHi in HCO(3-)-buffered media was lower than in the absence of HCO3- (7.35 vs. 7.80). pHi recovery from an acid load in HCO3- was Na- dependent but was inhibited only 18% by 1 mM amiloride. The amiloride-independent pHi recovery was inhibited 49% by pre-incubation of cells in 5 mM DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). These data suggest that Caco-2 cells possess: (a) both apical and basolateral membrane Na(+)-H+ exchange mechanisms, the apical exchanger being relatively resistant to amiloride, similar to apical Na(+)-H+ exchangers in several normal epithelia; and (b) a Na(-)-dependent HCO3- transport system, either Na(+)-HCO3- cotransport or Na(-)-dependent Cl(-)-HCO3- exchange.
完整肠上皮细胞中的酸碱转运系统尚未完全明确。因此，我们研究了人细胞系Caco-2，该细胞系培养在聚四氟乙烯膜上，形成融合单层，在透射电子显微镜下可见顶端微绒毛，且微绒毛水解酶逐渐富集。将负载有pH敏感染料BCECF-AM（2'，7'-双（羧乙基）-5-羧基荧光素）的单层细胞（16至25日龄）置于荧光分光光度计比色皿中，以便用Hepes或HCO₃⁻缓冲培养基选择性地灌流顶端和基底外侧表面。通过双激发荧光分光光度法测量细胞内pH（pHi）；使用含有尼日利亚菌素和110 mM K⁺的标准溶液进行校准，该标准溶液对应于Caco-2细胞单层中测得的细胞内[K⁺]。在无HCO₃⁻（Hepes缓冲）培养基中，用1 mM氨氯吡脒或无Na⁺培养基双侧灌流分别可逆地抑制了细胞内酸负荷（NH₄Cl脉冲）后pHi的恢复，抑制率分别为86%和98%。向顶端或基底外侧灌流液中选择性地重新添加Na⁺也诱导了pHi的恢复，同侧氨氯吡脒（1 mM）可抑制该恢复，而异侧则无抑制作用。顶端重新添加Na⁺诱导的pHi恢复对Na⁺的米氏常数（Km）为30 mM，对氨氯吡脒的抑制常数（Ki）相对较高，为45.5 μM。HCO₃⁻缓冲培养基中的初始pHi低于无HCO₃⁻时（7.35对7.80）。HCO₃⁻中酸负荷后的pHi恢复依赖于Na⁺，但1 mM氨氯吡脒仅抑制18%。细胞在5 mM DIDS（4,4'-二异硫氰酸根合芪-2,2'-二磺酸）中预孵育后，不依赖氨氯吡脒的pHi恢复被抑制49%。这些数据表明，Caco-2细胞具有：（a）顶端和基底外侧膜的Na⁺-H⁺交换机制，顶端交换器对氨氯吡脒相对耐药，类似于几种正常上皮细胞中的顶端Na⁺-H⁺交换器；（b）一种依赖Na⁺的HCO₃⁻转运系统，要么是Na⁺-HCO₃⁻共转运，要么是依赖Na⁺的Cl⁻-HCO₃⁻交换。
