Abstract
The H-K-ATPase (HKA), a potassium-dependent proton transporter in the outer medullary collecting duct (OMCD) plays an important role in acid-base homeostasis. The OMCD contains two HKA isoforms; gastric (HKAα1), dominant under normal dietary conditions (ND), and colonic (HKAα2), induced under a K-free diet (KD). The enzymatic activity (EA) of HKA in the OMCD is incompletely understood. The focus of the present study is elucidating the EA of the HKA in HKAα1 and HKAα2 knockout (KO) mice under ND and KD. KO mice were subjected to ND or KD for 10 days. Ten OMCD tubules were extracted, half placed in potassium-free media (Solution 2), half in potassium-containing media (Solution 3). Fluorescence measurements are based on the hydrolysis of ATP to ADP, coupled with the oxidation of NADH. ADP is determined by a decrease in NADH fluorescence. In K presence, NADH fluorescence of HKAα1 KO mice read 13.5 ± 0.7 ppm for ND and 10.3 ± 0.2 ppm for KD, indicating stimulation of the colonic isoform. HKAα2 KO mice averaged 6.8 ± 0.3 ppm for ND and 5.4 ± 0.3 ppm for KD in solution 2 (p p α2 isoform. A significant difference in ATP production in HKAα2 KO mice is likely due to enhanced EA of H-ATPase under potassium depletion.
Highlights
The outer medullary collecting duct (OMCD) plays an important role in acid-base homeostasis by mediating transepithelial bicarbonate transport and urinary acidification
The experiment groups of mice we selected were HKAα1 KO and HKAα2 KO. These two types of transgenic mice were under normal dietary conditions (ND) or K-free diet (KD) diet
The ADP generated in the assay system is determined by a decrease in NADH fluorescence
Summary
The outer medullary collecting duct (OMCD) plays an important role in acid-base homeostasis by mediating transepithelial bicarbonate transport and urinary acidification. Bicarbonate absorption by type A intercalated cells (ICs) in the OMCD segment is thought to be mediated by an apical H-K-ATPase coupled to a basolateral Chloride-Bicarbonate exchanger (AE1) [1]. The evidence for a predominant role of H-K-ATPase(s) in luminal acidification is provided by studies that examine the effect of pharmacological agents and luminal K removal on the rate of net bicarbonate absorption (JtCO2) in the OMCD. Our previous studies indicate the presence of at least two H-K-ATPase (HK) isoforms, HKAα1 (gastric) and HKAα2 (colonic), in OMCD cells. We demonstrated active proton secretion (JtCO2), one-third of which was blocked by bafilomycin, a specific inhibitor of the H-ATPase. Molecular studies have demonstrated the third H-K-ATPase isoform (HKβ) in the kidney. It has been reported that HKβ subunit is required for acid-secretory activity of parietal cells in vivo [2] [3]
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