Ammonium transport by the colonic H+-K+-ATPase expressed inXenopusoocytes (original) (raw)
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Journal of Biological Chemistry, 1999
Recent studies have suggested that the colonic H ؉ ,K ؉-ATPase (HK␣ 2) can secrete either Na ؉ or H ؉ in exchange for K ؉. If correct, this view would indicate that the transporter could function as either a Na ؉ or a H ؉ pump. To investigate this possibility a series of experiments was performed using apical membranes from rat colon which were enriched in colonic H ؉ ,K ؉-ATPase protein. An antibody specific for HK␣ 2 was employed to determine whether HK␣ 2 functions under physiological conditions as a Na ؉-dependent or Na ؉-independent K ؉-ATPase in this same membrane fraction. K ؉-ATPase activity was measured as [␥-32 P]ATP hydrolysis. The Na ؉dependent K ؉-ATPase accounted for approximately 80% of overall K ؉-ATPase activity and was characterized by insensitivity to Sch-28080 but partial sensitivity to ouabain. The Na ؉-independent K ؉-ATPase activity was insensitive to both Sch-28080 and ouabain. Both types of K ؉-ATPase activity substituted NH 4 ؉ for K ؉ in a similar manner. Furthermore, our results demonstrate that when incubated with native distal colon membranes, the blocking antibody inhibited dramatically Na ؉-dependent K ؉-ATPase activity. Therefore, these data demonstrate that HK␣ 2 can function in native distal colon apical membranes as a Na ؉-dependent K ؉-ATPase. Elucidation of the role of the pump as a transporter of Na ؉ versus H ؉ or NH 4 ϩ versus K ؉ in vivo will require additional studies.
Does the colonic H,K-ATPase also act as an Na,K-ATPase?
Proceedings of the National Academy of Sciences, 1998
We previously have demonstrated that the colonic P-ATPase α subunit cDNA encodes an H,K-ATPase when expressed in Xenopus laevis oocytes. Besides its high level of amino acid homology (75%) with the Na,K-ATPase, the colonic H,K-ATPase also shares a common pharmacological profile with Na,K-ATPase, because both are ouabain-sensitive and Sch 28080-insensitive. These features raise the possibility that an unrecognized property of the colonic H,K-ATPase would be Na + translocation. To test this hypothesis, ion-selective microelectrodes were used to measure the intracellular Na + activity of X. laevis oocytes expressing various combinations of P-ATPase subunits. The results show that expression in oocytes of the colonic H,K-ATPase affects intracellular Na + homeostasis in a way similar to the expression of the Bufo marinus Na,K-ATPase; intracellular Na + activity is lower in oocytes expressing the colonic H,K-ATPase or the B. marinus Na,K-ATPase than in oocytes expressing the gastric H,K-A...
Ammonium transport in the colonic crypt cell line, T84: role for Rhesus glycoproteins and NKCC1
American Journal of Physiology-Gastrointestinal and Liver Physiology, 2007
Although colonic lumen NH4+levels are high, 15–44 mM normal range in humans, relatively few studies have addressed the transport mechanisms for NH4+. More extensive studies have elucidated the transport of NH4+in the kidney collecting duct, which involves a number of transporter processes also present in the distal colon. Similar to NH4+secretion in the renal collecting duct, we show that the distal colon secretory model, T84 cell line, has the capacity to secrete NH4+and maintain an apical-to-basolateral NH4+gradient. NH4+transport in the secretory direction was supported by basolateral NH4+loading on NKCC1, Na+-K+-ATPase, and the NH4+transporter, RhBG. NH4+was transported on NKCC1 in T84 cells nearly as well as K+as determined by bumetanide-sensitive86Rb-uptake.86Rb-uptake and ouabain-sensitive current measurement indicated that NH4+is transported by Na+-K+-ATPase in these cells to an equal extent as K+. T84 cells expressed mRNA for the basolateral NH4+transporter RhBG and the api...
Ouabain-sensitive H,K-ATPase Functions as Na,K-ATPase in Apical Membranes of Rat Distal Colon
Journal of Biological Chemistry, 2000
Na,K-ATPase activity has been identified in the apical membrane of rat distal colon, whereas ouabain-sensitive and ouabain-insensitive H,K-ATPase activities are localized solely to apical membranes. This study was designed to determine whether apical membrane Na,K-ATPase represented contamination of basolateral membranes or an alternate mode of H,K-ATPase expression. An antibody directed against the H,K-ATPase ␣ subunit (HKc␣) inhibited apical Na,K-ATPase activity by 92% but did not alter basolateral membrane Na,K-ATPase activity. Two distinct H,K-ATPase isoforms exist; one of which, the ouabain-insensitive HKc␣, has been cloned. Because dietary sodium depletion markedly increases ouabain-insensitive active potassium absorption and HKc␣ mRNA and protein expression, Na,K-ATPase and H,K-ATPase activities and protein expression were determined in apical membranes from control and sodium-depleted rats. Sodium depletion substantially increased ouabain-insensitive H,K-ATPase activity and HKc␣ protein expression by 109 -250% but increased ouabain-sensitive Na,K-ATPase and H,K-ATPase activities by only 30% and 42%, respectively. These studies suggest that apical membrane Na,K-ATPase activity is an alternate mode of ouabain-sensitive H,K-ATPase and does not solely represent basolateral membrane contamination. In previous studies dietary sodium depletion and aldosterone infusion via mini-pumps produced identical changes of both sodium, chloride, and potassium transport in rat distal colon (34 -36). In addition, serum aldosterone levels were similar in the dietary sodium-depleted animals and in those that were infused with aldosterone via minipumps (37). Thus, in the present manuscript, aldosterone is at times used to refer to sodium-depleted animals.
Journal of Biological Chemistry, 1998
demonstrated that the ␣-subunit of the colonic H ؉ ,K ؉ -ATPase (HK␣ 2 ) requires coexpression with a -subunit to support H ؉ /K ؉ transport in a heterologous expression system (Xenopus laevis oocytes). In these studies, HK␣ 2 formed stable and functional ␣⅐ complexes when coexpressed with either the rat  1 -subunit of the Na ؉ ,K ؉ -ATPase or the -subunit of the gastric H ؉ ,K ؉ -ATPase, suggesting that different -subunits may interact with HK␣ 2 . The present studies tested this hypothesis by development and application of a specific antibody against HK␣ 2 peptide. Subsequently, immunoprecipitation experiments were performed to determine if HK␣ 2 co-precipitates with the same -subunit in organs known to express HK␣ 2 protein. The data demonstrate that HK␣ 2 assembles with  1 -Na ؉ ,K ؉ -ATPase in the renal medulla and in distal colon.
The α-Subunit of the Colonic H+,K+-ATPase Assembles with β1-Na+,K+-ATPase in Kidney and Distal Colon
Journal of Biological Chemistry, 1998
demonstrated that the ␣-subunit of the colonic H ؉ ,K ؉-ATPase (HK␣ 2) requires coexpression with a -subunit to support H ؉ /K ؉ transport in a heterologous expression system (Xenopus laevis oocytes). In these studies, HK␣ 2 formed stable and functional ␣⅐ complexes when coexpressed with either the rat  1-subunit of the Na ؉ ,K ؉-ATPase or the -subunit of the gastric H ؉ ,K ؉-ATPase, suggesting that different -subunits may interact with HK␣ 2. The present studies tested this hypothesis by development and application of a specific antibody against HK␣ 2 peptide. Subsequently, immunoprecipitation experiments were performed to determine if HK␣ 2 co-precipitates with the same -subunit in organs known to express HK␣ 2 protein. The data demonstrate that HK␣ 2 assembles with  1-Na ؉ ,K ؉-ATPase in the renal medulla and in distal colon.
Regulation of the Na + 2Cl - K + cotransporter in isolated rat colon crypts
Pflügers Archiv: European Journal of Physiology, 2000
The Na + 2Cl-K + cotransporter accepts NH 4 + at its K +-binding site. Therefore, the rate of cytosolic acidification after NH 4 + addition to the bath (20 mmol/l) measured by BCECF fluorescence can be used to quantify the rate of this cotransporter. In isolated colon crypts of rat distal colon (RCC) addition of NH 4 + led to an initial alkalinization, corresponding to NH 3 uptake. This was followed by an acidification, corresponding to NH 4 + uptake. The rate of this uptake was quantified by exponential curve fitting and is given in arbitrary units (∆ fluorescence ratio units/1000 s). In pilot experiments it was shown that the pH signal caused by the Na + 2Cl-K + cotransporter could be amplified if the experiments were carried out in the presence of bath Ba 2+ to inhibit NH 4 + uptake via K + channels. Therefore all subsequent experiments were performed in the presence of 1 mmol/l Ba 2+. In the absence of any secretagogue, preincubation of RCC in a low-Clsolution (4 mmol/l) for 10 min enhanced the uptake rate significantly from 1.70±0.11 to 2.54±0.27 U/1000 s (n=20). The addition of 100 mmol/l mannitol (hypertonic solution) enhanced the rate significantly from 1.93±0.17 to 2.84±0.43 U/1000 s (n=5). Stimulation of NaCl secretion by a solution containing 100 µmol/l carbachol (CCH) led to a small but significant increase in NH 4 + uptake rate from 2.06±0.34 to 2.40±0.30 U/1000 s (n=11). The increase in uptake rate observed with stimulation of the cAMP pathway by isobutylmethylxanthine (IBMX) and forskolin (100 µmol/l and 5 µmol/l, respectively) was from 2.39±0.24 to 3.06±0.36 U/1000 s (n=24). Whatever the mechanism used to increase the NH 4 + uptake rate, azosemide (500 µmol/l) always reduced this rate to control values. Hence three manoeuvres enhanced loop-diuretic-inhibitable uptake rates of the Na + 2Cl-K + cotransporter: (1) lowering of cytosolic Clconcentration; (2) cell shrinkage ; (3) activation of NaCl secretion by carbachol and (4) activation of NaCl secretion by cAMP. The common denominator of all four activation pathways may be a transient fall in cell volume.
The Rat Distal Colon P-ATPase α Subunit Encodes a Ouabain-sensitive H+,K+-ATPase
Journal of Biological Chemistry, 1996
The functional properties and the pharmacological profile of the recently cloned cDNA colonic P-ATPase ␣ subunit (Crowson, M. S., and Shull, G. E. (1992) J. Biol. Chem. 267, 13740-13748) were investigated by using the Xenopus oocyte expression system. Xenopus oocytes were injected with ␣ subunit cRNAs from Bufo marinus bladder or rat distal colon and/or with  subunit cRNA from B. marinus bladder. Two days after injection, K ؉ uptake was measured by using 86 Rb ؉ as a K ؉ surrogate, and pH measurements were performed by means of ionselective microelectrodes. Co-injection of ␣ and  subunit cRNAs lead to a large increase in 86 Rb ؉ uptake, an intracellular alkalinization, and an extracellular medium acidification, as compared to ␣ or  injection alone. These results indicate that the colonic P-ATPase ␣ subunit, like the bladder ␣ subunit, acts as a functional H ؉ ,K ؉-ATPase, and that co-expression of ␣ and  subunits is required for the function. External K ؉ activation of the 86 Rb ؉ uptake had a K 1/2 ϳ 440 M for the bladder isoform (consistent with the previously reported value (Jaisser, F., Horisberger, J. D., Geering, K., and Rossier, B. C. (1933) J. Cell Biol. 123, 1421-1431)) and a K 1/2 ϳ 730 M for the colonic isoform. Sch28080 was ineffective to reduce 86 Rb ؉ uptake whereas ouabain inhibited the activity expressed from rat colon ␣ subunit with a K i of 970 M when measured at the V max of the enzyme. We conclude that, when expressed in Xenopus oocytes, the rat colon P-ATPase ␣ subunit encodes a ouabain-sensitive H ؉ ,K ؉-ATPase.