INTRACELLULAR-PH DEPENDENCE OF NA-H EXCHANGE AND ACID LOADING IN QUIESCENT AND ARGININE VASOPRESSIN-ACTIVATED MESANGIAL CELLS
INTRACELLULAR-PH DEPENDENCE OF NA-H EXCHANGE AND ACID LOADING IN QUIESCENT AND ARGININE VASOPRESSIN-ACTIVATED MESANGIAL CELLS
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DOI：
10.1073/pnas.87.15.5921
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发表时间：
1990-08-01
影响因子：
11.1
通讯作者：
BORON, WF
中科院分区：
文献类型：
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作者：
BOYARSKY, G;GANZ, MB;BORON, WF
我们用pH敏感染料2“，7”-双（2-羧乙基）-5（和-6）羧基荧光素研究了在无HCO 3-存在下单个系膜细胞（MC）的细胞内pH（pHi）调节。我们的方法是通过NH 4+预脉冲对细胞进行酸负荷，并监测随后的pHi恢复。先前对MCs和其他细胞的研究表明，在恢复开始前加入乙基异丙基阿米洛利（EIPA）或除去Na+会阻止恢复，这表明在低pH值下，只有Na-H交换有助于恢复。该结论通常外推至整个pHi范围。为了测试这一点，我们在不同的pHi值下用EIPA中断回收，发现EIPA暴露了背景酸化，该背景酸化在pHi小于约6.7时可忽略，但在更高的pHi值下急剧增加。校正该EIPA不敏感组分的总回收率，我们发现EIPA敏感（Na-H交换）率随着pHi在6.3和6.7之间的增加而急剧下降，但在6.7和7.2之间相对pHi不敏感。因此，当pHi接近约时，回收停止。7.2不是因为Na-H交换减慢，而是因为酸加载加速。应用促分裂原精氨酸加压素（AVP; 100 nM）引起pHi快速降低至约0.4，随后缓慢升高至比初始pHi高约0.15的水平。与pHi的这种双相变化相一致的是Na-H交换动力学的双相变化。在早期阶段（即，在加入AVP后约8分钟开始pHi恢复），AVP使交换剂的pHi依赖性线性化;在pHi小于约6.7时，其速率不受AVP影响，但在较高pHi值时逐渐受到抑制。在后期阶段（即，在加入AVP后约14分钟开始pHi恢复），AVP使这种线性pHi依赖性向碱性方向移动;交换器在pHi < 6.9时受到刺激，但在较高pHi值时受到适度抑制（即，在生理范围内）。 在所有时间，AVP大大抑制背景酸负荷。因此，AVP升高稳态pHi不是因为Na-H交换被刺激，而是因为尽管交换器被抑制，但酸加载被抑制得更多。
We studied intracellular pH (pHi) regulation in the absence of HCO3- in single mesangial cells (MCs) with the pH-sensitive dye 2'',7''-bis(2-carboxyethyl)-5(and -6)carboxyfluorescein. Our approach was to acid load the cells by an NH4+ prepulse and to monitor the subsequent pHi recovery. Previous work on MCs and other cells has shown that the recovery is prevented by adding ethylisopropyl amiloride (EIPA) or removing Na+ before the recovery begins, suggesting that at low pHi only Na-H exchange contributes to the recovery. This conclusion is often extrapolated to the entire pHi range. To test this, we interrupted the recovery with EIPA at various pHi values, finding that EIPA unmasked a background acidification that was negligible at pHi less than .apprxeq.6.7 but increased steeply at higher pHi values. Correcting the total recovery rate for this EIPA-insensitive component, we found that the EIPA-sensitive (Na-H exchange) rate fell steeply with increasing pHi between 6.3 and 6.7 but was relatively pHi insensitive between 6.7 and 7.2. Thus, the recovery halts as pHi approaches .apprxeq. 7.2 not so much because Na-H exchange slows, but because acid loading accelerates. Applying the mitogen arginine vasopressin (AVP; 100 nM) caused a rapid pHi decrease of .apprxeq.0.4, followed by a slower increase to a level .apprxeq.0.15 higher than the initial pHi. Coincident with this biphasic change in pHi was a biphasic change in Na-H exchange kinetics. In the early phase (i.e., pHi recovery commencing .apprxeq.8 min after AVP addition), AVP linearized the pHi dependence of the exchanger; its rate was unaffected by AVP at pHi less than .apprxeq.6.7 but was progressively inhibited at higher pHi values. In the later phase (i.e., pHi recovery commencing .apprxeq.14 min after AVP addition), AVP shifted this linear pHi dependence in the alkaline direction; the exchanger was stimulated at pHi < 6.9 but was modestly inhibited at higher pHi values (i.e., in the physiological range). At all times, AVP greatly inhibited background acid loading. Thus, AVP raises steady-state pHi not because Na-H exchange is stimulated but because, although the exchanger is inhibited, acid loading is inhibited even more.