Bleich M, Warth R, Thiele I, Greger R
Physiologisches Institut, Albert-Ludwigs-Universität, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany.
Pflugers Arch. 1998 Jul;436(2):248-54. doi: 10.1007/s004240050629.
Isolated in vitro perfused rectal gland tubules (RGT) were preincubated with the pH-sensitive dye 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and pH-regulatory mechanisms were studied. A reduction of bath Cl- concentration from 269 to 6 mmol/l increased the fluorescence ratio 488/436 [corresponding to cytosolic pH (pHi)] slightly but significantly (n=10). Depolarization by Ba2+ (1 mmol/l) or a bath solution containing 30 mmol/l K+ (n=4-6) increased the fluorescence ratio (pHi). These data suggest that HCO3- uptake and/or H+ extrusion is dependent on Cl- and/or voltage. A reduction of bath Na+ from 278 to 5 mmol/l reduced the ratio significantly (n=3). Addition of trimethylamine (Trima+, 20 mmol/l) alkalinized cytosolic pH (n=7). Similarly, addition of NH4+ (20 mmol/l) led to an initial alkalinization and a strong acidification when NH4+ was removed (n=59). The initial pHi-recovery rates after NH4+ removal were quantified and the responsible H+ extrusion and/or HCO3- import systems were examined. The recovery was almost completely abolished when the extracellular Na+ concentration was reduced to 5 mmol/l. In the presence of normal Na+, recovery was slower in the absence as compared to the presence of HCO3- (n=5). It was inhibited by 4, 4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) (0.5 mmol/l, n=11) in the presence of HCO3- and in the absence of HCO3- by the Na+/H+-exchange blocker HOE694 (0.5 mmol/l, n=6). These data suggest that acid extrusion probably occurs by basolateral Na+-2HCO3-/Cl- exchange in the presence of HCO3- and by basolateral Na+/H+ exchange in the absence of HCO3-. Luminal perfusion with a solution containing a low Cl- concentration (6 mmol/l) increased the fluorescence ratio (pHi) (n=5). The ratio (pHi) was further increased and pH recovery further delayed by basolateral addition of Trima+ (20 mmol/l, n=3). These data suggest that the HCO3-/Cl- exchanger is present in the luminal membrane. Luminal HCO3-/Cl- exchange and basolateral Na+-2HCO3-/Cl- exchange may work in tandem to secrete HCO3- and exchange it for luminal Cl-.
将离体的体外灌注直肠腺小管(RGT)与pH敏感染料2',7'-双(羧乙基)-5(6)-羧基荧光素(BCECF)进行预孵育,并研究pH调节机制。将浴液中Cl-浓度从269 mmol/l降至6 mmol/l,荧光比率488/436[对应于胞质pH(pHi)]略有但显著升高(n = 10)。Ba2+(1 mmol/l)或含30 mmol/l K+的浴液使膜去极化(n = 4 - 6),荧光比率(pHi)升高。这些数据表明,HCO3-摄取和/或H+排出依赖于Cl-和/或电压。将浴液中Na+从278 mmol/l降至5 mmol/l,显著降低了该比率(n = 3)。添加三甲胺(Trima+,20 mmol/l)使胞质pH碱化(n = 7)。同样,添加NH4+(20 mmol/l)导致初始碱化,去除NH4+时则导致强烈酸化(n = 59)。对去除NH4+后的初始pHi恢复速率进行了定量,并研究了相关的H+排出和/或HCO3-导入系统。当细胞外Na+浓度降至5 mmol/l时,恢复几乎完全消除。在正常Na+存在下,与存在HCO3-相比,不存在HCO3-时恢复较慢(n = 5)。在存在HCO3-时,它被4,4'-二异硫氰基芪-2,2'-二磺酸(DIDS)(0.5 mmol/l,n = 11)抑制,在不存在HCO3-时,被Na+/H+交换阻滞剂HOE694(0.5 mmol/l,n = 6)抑制。这些数据表明,在存在HCO3-时,酸排出可能通过基底外侧Na+-2HCO3-/Cl-交换发生,在不存在HCO3-时,通过基底外侧Na+/H+交换发生。用含低Cl-浓度(6 mmol/l)的溶液进行管腔灌注,荧光比率(pHi)升高(n = 5)。基底外侧添加Trima+(20 mmol/l,n = 3)进一步升高了该比率(pHi),并进一步延迟了pH恢复。这些数据表明,HCO3-/Cl-交换体存在于管腔膜中。管腔HCO3-/Cl-交换和基底外侧Na+-2HCO3-/Cl-交换可能协同作用以分泌HCO3-并将其与管腔Cl-进行交换。