Okada M, Saito Y, Sawada E, Nishiyama A
Department of Physiology, Tohoku University School of Medicine, Sendai, Japan.
Pflugers Arch. 1991 Oct;419(3-4):338-48. doi: 10.1007/BF00371116.
The mechanism of regulation of intracellular pH (pHi) in dispersed acini from the rat mandibular salivary gland has been studied with a microfluorimetric imaging method and the pH probe 2',7'-bis(2-carboxyethyl)-5(and -6)-carboxyfluorescein. The pHi in the TRIS/HEPES-buffered standard solution was 7.29 +/- 0.01. Addition of 1 mumol/l acetylcholine (ACh) or ionomycin caused a sustained increase in the pHi. These agents decreased pHi in the absence of external Na+ or in the presence of amiloride. The rate of pHi recovery from an acid load after NH+4 prepulse was a linear function of pHi and increased as pHi became more acidic. Addition of ACh shifted the relationship towards a more alkaline pHi range. The increase in pHi induced by ACh or ionomycin was not inhibited by the protein kinase C inhibitors staurosporine (10 nM) and 1-(5-isoquinolinesulfonyl)-1-methylpiperazine (50 mumol/l). Addition of 0.1-1 mumol/l phorbol 12-myristate 13-acetate (TPA) had little effect on pHi within 10 min; however, exposure to TPA for 120 min resulted in a significant rise in pHi. In Ca(2+)-free solution with 50 mumol/l 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate, the ACh-induced rise in both pHi and cytosolic Ca2+ concentration was suppressed. ACh and ionomycin caused an increment of amiloride-sensitive acid output into the extracellular fluid, while 20 mumol/l 1-oleoyl-2-acetylglycerol had little effect on it. It was concluded that (a) stimulation with ACh activated the Na+/H+ antiport in the plasma membrane, (b) ACh also stimulated the intracellular acid production but acid extrusion by the Na+/H+ antiport prevented the cell from intracellular acidification, and (c) the major route of signal transduction for the ACh-induced activation of the Na+/H+ antiport was independent of protein kinase C but was dependent on the rise in cytosolic Ca2+ concentration. The implication of the cytosolic acidification and cell volume change in pHi regulation is discussed.
采用显微荧光成像法和pH探针2',7'-双(2-羧乙基)-5(及-6)-羧基荧光素，对大鼠下颌唾液腺分散腺泡细胞内pH（pHi）的调节机制进行了研究。在TRIS/HEPES缓冲标准溶液中，pHi为7.29±0.01。添加1μmol/L乙酰胆碱（ACh）或离子霉素会导致pHi持续升高。在无细胞外Na+或存在氨氯吡咪的情况下，这些试剂会使pHi降低。NH+4预脉冲后酸负荷引起的pHi恢复速率是pHi的线性函数，且随着pHi变得更酸而增加。添加ACh会使这种关系向更碱性的pHi范围偏移。ACh或离子霉素诱导的pHi升高不受蛋白激酶C抑制剂星形孢菌素（10 nM）和1-(5-异喹啉磺酰基)-1-甲基哌嗪（50μmol/L）的抑制。添加0.1 - 1μmol/L佛波醇12-肉豆蔻酸酯13-乙酸酯（TPA）在10分钟内对pHi影响不大；然而，暴露于TPA 120分钟会导致pHi显著升高。在含有50μmol/L 8-(二乙氨基)-辛基-3,4,5-三甲氧基苯甲酸酯的无Ca(2+)溶液中，ACh诱导的pHi和胞质Ca2+浓度升高均受到抑制。ACh和离子霉素会使氨氯吡咪敏感的酸分泌到细胞外液中增加，而20μmol/L 1-油酰基-2-乙酰甘油对此影响不大。得出以下结论：(a) ACh刺激激活了质膜上的Na+/H+反向转运体；(b) ACh还刺激了细胞内酸的产生，但Na+/H+反向转运体的酸外排阻止了细胞内酸化；(c) ACh诱导的Na+/H+反向转运体激活的主要信号转导途径独立于蛋白激酶C，但依赖于胞质Ca2+浓度的升高。文中讨论了胞质酸化和细胞体积变化在pHi调节中的意义。
