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), domin...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.展开更多
H+-K+-ATPase (HKA) is composed of two different subunits: an alpha and a beta subunit. Previous studies have shown that in the kidney gastric HKA (HKA alpha 1) predominates under normal dietary conditions while coloni...H+-K+-ATPase (HKA) is composed of two different subunits: an alpha and a beta subunit. Previous studies have shown that in the kidney gastric HKA (HKA alpha 1) predominates under normal dietary conditions while colonic HKA (HKA alpha 2) predominates under potassium depleted conditions [1]. The purpose of the current study was to elucidate the association between the beta and different alpha subunits from stomach, colon and kidney under normal and potassium depleted conditions. Black Swiss mice were fed a potassium-free diet for 2 weeks, beta subunit expression of HKA in stomach mucosae, colon mucosae and renal outer medulla was examined and compared between normal diet and potassium depleted diet. In wild type (WT) mice, the concentrations of the beta subunit under potassium deficient conditions were found significantly increased compared with normal dietary conditions in colon mucosae (8.27 ± 0.73 vs 6.62 ± 0.55 μg/μl, n = 7, p = 0.0416), whereas in cHKA (HKA alpha 2) mice colon mucosae, the concentrations of the beta subunit were statistically the same (5.05 ± 0.31 vs 4.76 ± 0.37 μg/μl, n = 13, p = 0.2833), and the concentration of the beta subunit stayed the same in renal outer medulla and stomach mucosae as well. The data indicate that potassium deficiency results in a significant increase in the levels of HKA beta subunit concentration in the colonic tissue of WT mice. The results indicate that the HKA beta subunit associates with the cHKA (HKA alpha 2) in order to mediate bicarbonate reabsorption under potassium depletion (hypokalemia)展开更多
文摘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.
文摘H+-K+-ATPase (HKA) is composed of two different subunits: an alpha and a beta subunit. Previous studies have shown that in the kidney gastric HKA (HKA alpha 1) predominates under normal dietary conditions while colonic HKA (HKA alpha 2) predominates under potassium depleted conditions [1]. The purpose of the current study was to elucidate the association between the beta and different alpha subunits from stomach, colon and kidney under normal and potassium depleted conditions. Black Swiss mice were fed a potassium-free diet for 2 weeks, beta subunit expression of HKA in stomach mucosae, colon mucosae and renal outer medulla was examined and compared between normal diet and potassium depleted diet. In wild type (WT) mice, the concentrations of the beta subunit under potassium deficient conditions were found significantly increased compared with normal dietary conditions in colon mucosae (8.27 ± 0.73 vs 6.62 ± 0.55 μg/μl, n = 7, p = 0.0416), whereas in cHKA (HKA alpha 2) mice colon mucosae, the concentrations of the beta subunit were statistically the same (5.05 ± 0.31 vs 4.76 ± 0.37 μg/μl, n = 13, p = 0.2833), and the concentration of the beta subunit stayed the same in renal outer medulla and stomach mucosae as well. The data indicate that potassium deficiency results in a significant increase in the levels of HKA beta subunit concentration in the colonic tissue of WT mice. The results indicate that the HKA beta subunit associates with the cHKA (HKA alpha 2) in order to mediate bicarbonate reabsorption under potassium depletion (hypokalemia)
