Ravesloot J H, Eisen T, Baron R, Boron W F
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
J Gen Physiol. 1995 Feb;105(2):177-208. doi: 10.1085/jgp.105.2.177.
Osteoclasts resorb bone by pumping of H+ into a compartment between the cell and the bone surface. Intracellular pH (pHi) homeostasis requires that this acid extrusion, mediated by a vacuolar-type H+ ATPase, be complemented by other acid-base transporters. We investigated acid-extrusion mechanisms of single, freshly isolated, neonatal rat osteoclasts. Cells adherent to glass coverslips were studied in the nominal absence of CO2/HCO3-, using the pH-sensitive dye BCECF and a digital imaging system. Initial pHi averaged 7.31 and was uniform throughout individual cells. Intrinsic buffering power (beta 1) decreased curvilinearly from approximately 25 mM at pHi = 6.4 to approximately 6.0 mM at pHi = 7.4. In all polygonally shaped osteoclasts, and approximately 60% of round osteoclasts (approximately 20% of total), pHi recovery from acid loads was mediated exclusively by Na-H exchange. In these pattern-1 cells, pHi recovery was 95% complete within 200 s, and was blocked by removing Na+, or by applying 1 mM amiloride, 50 microM ethylisopropylamiloride (EIPA), or 50 microM hexamethyleneamiloride (HMA). The apparent K1/2 for HMA ([Na+]o = 150 mM) was 49 nM, and the apparent K1/2 for Na+ was 45 mM. Na-H exchange, corrected for amiloride-insensitive fluxes, was half maximal at pHi 6.73, with an apparent Hill coefficient for intracellular H+ of 2.9. Maximal Na-H exchange averaged 741 microM/s. In the remaining approximately 40% of round osteoclasts (pattern-2 cells), pHi recovery from acid loads was brisk even in the absence of Na+ or presence of amiloride. This Na(+)-independent pHi recovery was blocked by 7-chloro-4-nitrobenz-2-oxa-1,3-diazol (NBD-Cl), a vacuolar-type H+ pump inhibitor. Corrected for NBD-Cl insensitive fluxes, H+ pump fluxes decreased approximately linearly from 96 at pHi 6.8 to 11 microM/s at pHi 7.45. In approximately 45% of pattern-2 cells, Na+ readdition elicited a further pHi recovery, suggesting that H+ pumps and Na-H exchangers can exist simultaneously. We conclude that, under the conditions of our study, most neonatal rat osteoclasts express Na-H exchangers that are probably of the ubiquitous basolateral subtype. Some cells express vacuolar-type H+ pumps in their plasma membrane, as do active osteoclasts in situ.
破骨细胞通过将氢离子泵入细胞与骨表面之间的腔室来吸收骨组织。细胞内pH值（pHi）的稳态要求这种由液泡型氢离子ATP酶介导的酸排出，需由其他酸碱转运体来补充。我们研究了新生大鼠单个新鲜分离破骨细胞的酸排出机制。使用pH敏感染料BCECF和数字成像系统，在名义上不存在二氧化碳/碳酸氢根的条件下，对附着在玻璃盖玻片上的细胞进行研究。初始pHi平均为7.31，且在单个细胞内是均匀的。固有缓冲能力（β1）从pHi = 6.4时的约25 mM呈曲线下降至pHi = 7.4时的约6.0 mM。在所有多边形破骨细胞以及约60%的圆形破骨细胞（约占总数的20%）中，酸负荷后的pHi恢复完全由钠-氢交换介导。在这些模式1细胞中，pHi恢复在200秒内95%完成，并通过去除钠离子或应用1 mM氨氯吡脒、50 μM乙基异丙基氨氯吡脒（EIPA）或50 μM六亚甲基氨氯吡脒（HMA）而被阻断。HMA（[钠离子]o = 150 mM）的表观K1/2为49 nM，钠离子的表观K1/2为45 mM。校正氨氯吡脒不敏感通量后的钠-氢交换，在pHi 6.73时达到最大值的一半，细胞内氢离子的表观希尔系数为2.9。最大钠-氢交换平均为741 μM/秒。在其余约40%的圆形破骨细胞（模式2细胞）中，即使在没有钠离子或存在氨氯吡脒的情况下，酸负荷后的pHi恢复也很快。这种不依赖钠离子的pHi恢复被液泡型氢离子泵抑制剂7-氯-4-硝基苯并-2-恶唑-1,3-二氮杂环戊二烯（NBD-Cl）阻断。校正NBD-Cl不敏感通量后，氢离子泵通量从pHi 6.8时的96大致呈线性下降至pHi 7.45时的11 μM/秒。在约45%的模式2细胞中，重新添加钠离子会引起进一步的pHi恢复，这表明氢离子泵和钠-氢交换体可以同时存在。我们得出结论，在我们的研究条件下，大多数新生大鼠破骨细胞表达的钠-氢交换体可能是普遍存在的基底外侧亚型。一些细胞在其质膜中表达液泡型氢离子泵，原位的活跃破骨细胞也是如此。
