Siebens A W, Boron W F
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Gen Physiol. 1987 Dec;90(6):799-831. doi: 10.1085/jgp.90.6.799.
We used microelectrodes to examine the effects of organic substrates, particularly lactate (Lac-), on the intracellular pH (pHi) and basolateral membrane potential (Vbl) in isolated, perfused proximal tubules of the tiger salamander. Exposure of the luminal and basolateral membranes to 3.6 mM Lac- caused pHi to increase by approximately 0.2, opposite to the decrease expected from nonionic diffusion of lactic acid (HLac) into the cell. Addition of Lac- to only the lumen also caused alkalinization, but only if Na+ was present. This alkalinization was not accompanied by immediate Vbl changes, which suggests that it involves luminal, electroneutral Na/Lac cotransport. Addition of Lac- to only the basolateral solution caused pHi to decrease by approximately 0.08. The initial rate of this acidification was a saturable function of [Lac-], was not affected by removal of Na+, and was reversibly reduced by alpha-cyano-4-hydroxycinnamate (CHC). Thus, the pHi decrease induced by basolateral Lac- appears to be due to the basolateral entry of H+ and Lac-, mediated by an H/Lac cotransporter (or a Lac-base exchanger). Our data suggest that this transporter is electroneutral and is not present at the luminal membrane. A key question is how the addition of Lac- to the lumen increases pHi. We found that inhibition of basolateral H/Lac cotransport by basolateral CHC reduced the initial rate of pHi increase caused by luminal Lac-. On the other hand, luminal CHC had no effect on the luminal Lac(-)-induced alkalinization. These data suggest that when Lac- is present in the lumen, it enters the cell from the lumen via electroneutral Na/Lac cotransport and then exists with H+ across the basolateral membrane via electroneutral H/Lac cotransport. The net effect is transepithelial Lac- reabsorption, basolateral acid extrusion, and intracellular alkalinization.
我们使用微电极来研究有机底物，特别是乳酸（Lac-），对虎螈分离的灌注近端小管细胞内pH值（pHi）和基底外侧膜电位（Vbl）的影响。将管腔膜和基底外侧膜暴露于3.6 mM的Lac-中会使pHi升高约0.2，这与乳酸（HLac）通过非离子扩散进入细胞所预期的降低相反。仅向管腔中添加Lac-也会导致碱化，但前提是存在Na+。这种碱化并不伴随着Vbl的立即变化，这表明它涉及管腔的电中性Na/Lac协同转运。仅向基底外侧溶液中添加Lac-会使pHi降低约0.08。这种酸化的初始速率是[Lac-]的饱和函数，不受Na+去除的影响，并被α-氰基-4-羟基肉桂酸（CHC）可逆性降低。因此，基底外侧Lac-诱导的pHi降低似乎是由于H+和Lac-通过H/Lac协同转运体（或Lac-碱交换体）介导的基底外侧进入。我们的数据表明，这种转运体是电中性的，且不存在于管腔膜上。一个关键问题是向管腔中添加Lac-如何增加pHi。我们发现，基底外侧CHC对基底外侧H/Lac协同转运的抑制降低了管腔Lac-引起的pHi升高的初始速率。另一方面，管腔CHC对管腔Lac(-)诱导的碱化没有影响。这些数据表明，当Lac-存在于管腔中时，它通过电中性Na/Lac协同转运从管腔进入细胞，然后通过电中性H/Lac协同转运与H+一起穿过基底外侧膜。净效应是跨上皮Lac-重吸收、基底外侧酸排出和细胞内碱化。
