Paunescu Teodor G, Russo Leileata M, Da Silva Nicolas, Kovacikova Jana, Mohebbi Nilufar, Van Hoek Alfred N, McKee Mary, Wagner Carsten A, Breton Sylvie, Brown Dennis
Div. of Nephrology, Massachusetts General Hospital, 185 Cambridge St., CPZN 8150, Boston, MA 02114, USA.
Am J Physiol Renal Physiol. 2007 Dec;293(6):F1915-26. doi: 10.1152/ajprenal.00160.2007. Epub 2007 Sep 26.
Mice deficient in the ATP6V1B1 ("B1") subunit of the vacuolar proton-pumping ATPase (V-ATPase) maintain body acid-base homeostasis under normal conditions, but not when exposed to an acid load. Here, compensatory mechanisms involving the alternate ATP6V1B2 ("B2") isoform were examined to explain the persistence of baseline pH regulation in these animals. By immunocytochemistry, the mean pixel intensity of apical B2 immunostaining in medullary A intercalated cells (A-ICs) was twofold greater in B1-/- mice than in B1+/+ animals, and B2 was colocalized with other V-ATPase subunits. No significant upregulation of B2 mRNA or protein expression was detected in B1-/- mice compared with wild-type controls. We conclude that increased apical B2 staining is due to relocalization of B2-containing V-ATPase complexes from the cytosol to the plasma membrane. Recycling of B2-containing holoenzymes between these domains was confirmed by the intracellular accumulation of B1-deficient V-ATPases in response to the microtubule-disrupting drug colchicine. V-ATPase membrane expression is further supported by the presence of "rod-shaped" intramembranous particles seen by freeze fracture microscopy in apical membranes of normal and B1-deficient A-ICs. Intracellular pH recovery assays show that significant (28-40% of normal) V-ATPase function is preserved in medullary ICs from B1-/- mice. We conclude that the activity of apical B2-containing V-ATPase holoenzymes in A-ICs is sufficient to maintain baseline acid-base homeostasis in B1-deficient mice. However, our results show no increase in cell surface V-ATPase activity in response to metabolic acidosis in ICs from these animals, consistent with their inability to appropriately acidify their urine under these conditions.
液泡质子泵ATP酶（V-ATP酶）的ATP6V1B1（“B1”）亚基缺陷的小鼠在正常条件下可维持机体酸碱平衡，但在承受酸负荷时则不能。在此，我们研究了涉及交替ATP6V1B2（“B2”）同工型的代偿机制，以解释这些动物中基线pH调节的持续性。通过免疫细胞化学方法，在髓质A型闰细胞（A-ICs）中，B1基因敲除小鼠顶端B2免疫染色的平均像素强度比B1基因野生型动物高两倍，且B2与其他V-ATP酶亚基共定位。与野生型对照相比，在B1基因敲除小鼠中未检测到B2 mRNA或蛋白表达的显著上调。我们得出结论，顶端B2染色增加是由于含B2的V-ATP酶复合物从胞质溶胶重新定位到质膜。通过微管破坏药物秋水仙碱处理后B1缺陷型V-ATP酶在细胞内的积累，证实了含B2的全酶在这些结构域之间的循环利用。正常和B1缺陷型A-ICs顶端膜的冷冻断裂显微镜观察发现的“杆状”膜内颗粒，进一步支持了V-ATP酶的膜表达。细胞内pH恢复试验表明，B1基因敲除小鼠髓质ICs中保留了显著（正常的28 - 40%）的V-ATP酶功能。我们得出结论，A-ICs中含顶端B2的V-ATP酶全酶的活性足以维持B1缺陷小鼠的基线酸碱平衡。然而，我们的结果显示，这些动物的ICs中，响应代谢性酸中毒时细胞表面V-ATP酶活性没有增加，这与它们在这些条件下不能适当酸化尿液一致。
