Division of Nephrology, Hypertension and Transplantation, University of Florida College of Medicine, Gainesville 32610; and Gainesville Veterans Affairs Medical Center, Gainesville, Florida 32608
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ABSTRACT |
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The inner stripe of outer medullary collecting duct (OMCDis) is unique among collecting duct segments because both intercalated cells and principal cells secrete protons and reabsorb luminal bicarbonate. The current study characterized the mechanisms of OMCDis proton secretion. We used in vitro microperfusion, and we separately studied the principal cell and intercalated cell using differential uptake of the fluorescent, pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Both the principal cell and intercalated cell secreted protons, as identified as Na+/H+ exchange-independent intracellular pH (pHi) recovery from an intracellular acid load. Two proton transport activities were identified in the principal cell; one was luminal potassium dependent and Sch-28080 sensitive and the other was luminal potassium independent and luminal bafilomycin A1 sensitive. Thus the OMCDis principal cell expresses both apical H+-K+-ATPase and H+-ATPase activity. Intercalated cell Na+/H+ exchange-independent pHi recovery was approximately twice that of the principal cell and was mediated by pharmacologically similar mechanisms. We conclude 1) the OMCDis principal cell may contribute to both luminal potassium reabsorption and urinary acidification, roles fundamentally different from those of the principal cell in the cortical collecting duct; and 2) the OMCDis intercalated cell proton transporters are functionally similar to those in the principal cell, raising the possibility that an H+-K+-ATPase similar to the one present in the principal cell may contribute to intercalated cell proton secretion.
intracellular pH; 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein; intercalated cell; proton-adenosinetriphosphatase; proton-potassium-adenosinetriphosphatase; Sch-28080; bafilomycin A1
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INTRODUCTION |
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IN GENERAL, collecting duct principal cells are modeled to reabsorb sodium and secrete potassium, but not to secrete protons, whereas intercalated cells are modeled to transport protons and bicarbonate, but not to reabsorb sodium or secrete potassium. However, the principal cell in the inner stripe of the outer medullary collecting duct (OMCDis) appears to differ from other collecting duct principal cells. Under basal conditions, the OMCDis does not reabsorb sodium or secrete potassium, suggesting that neither the principal cell nor the intercalated cell reabsorbs potassium (36). At the single cell level, the OMCDis principal cell has a large apical fractional resistance (21), which suggests that apical sodium or potassium channels are either not present or not open under basal conditions. Thus, in contrast to the cortical collecting duct (CCD) principal cell, the OMCDis principal cell does not appear to either reabsorb sodium or secrete potassium.
One possible role for the OMCDis
principal cell is urinary acidification and luminal bicarbonate
reabsorption. Incubating in vitro microperfused
OMCDis with luminal
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)
acetoxymethyl ester (BCECF-AM), a lipid-permeable, nonfluorescent precursor of BCECF, identifies two cell populations (44) corresponding to the intercalated cell and principal cell populations. Studies using
this technique have shown that the
OMCDis principal cell exhibits
both sodium-independent proton secretion and basolateral Cl/HCO
3
exchange (44). Thus this cell appears to play a role in urinary
acidification and luminal bicarbonate reabsorption.
The mechanism of OMCDis principal
cell proton secretion is unclear. The two major mechanisms of
OMCDis proton secretion are an
apical H+-ATPase and an apical
H+-K+-ATPase
(4, 38). Several studies have examined the expression of
H+-ATPase protein,
HK1
protein1
and mRNA, and HK
2 mRNA in the
OMCDis, and none have shown
expression in the principal cell (1-3, 6, 8, 48). However, our
previous studies demonstrate the mRNA for the HK
subunit is
expressed in the OMCDis principal
cell (11), which suggests that this subunit may associate with a novel
-subunit in the principal cell.
The current study was designed to identify the mechanism(s) of OMCDis principal cell proton secretion and compare these mechanisms with those present in the intercalated cell. We microperfused OMCDis in vitro and identified intercalated cells and principal cells separately, based on the differential uptake of the fluorescent, pH-sensitive dye, BCECF (44). We measured proton secretion as the rate of intracellular pH (pHi) recovery from an intracellular acid load. Because basolateral Na+/H+ exchange is the primary pHi regulator in the OMCDis (18, 44), we inhibited this transporter to measure apical proton transporters. By using ionic substitutions and adding specific proton transporter inhibitors to the luminal fluid, we functionally identified the apical proton transporters present in the OMCDis principal cell and the intercalated cell.
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METHODS |
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Microperfusion. We used in vitro microperfusion techniques that we have previously described in detail (43, 44). Rabbits were injected with furosemide (10 mg im) 30 min before death to assist in dissection of viable tubules (44). The solutions used were artificial solutions, and, unless otherwise mentioned, contained (in mM), 119.2 NaCl, 3 KCl, 25 HEPES, 2 KH2PO4, 1 sodium acetate, 1.2 CaCl2, 1 MgSO4, 5 alanine, and 8.3 glucose. Potassium-free solutions substituted sodium for potassium. Ammonium chloride-containing solutions substituted NH4Cl for NaCl. All solutions were titrated to pH 7.40 with tetramethylammonium hydroxide, were gassed with 100% O2, and had osmolality adjusted to 290 ± 7 mosmol/kgH2O with NaCl. Tubules were bathed and perfused in a low-volume laminar flow chamber. Solutions were continuously bubbled with 100% O2 and delivered to the bathing chamber in water-jacketed lines at 37°C at a rate of ~6 ml/min.
pHi measurement. We measured OMCDis principal cell and intercalated cell pHi using techniques that we have described in detail previously (44). Briefly, BCECF-AM, 15 µM, was added to the luminal solution for ~5 min, resulting in heterogeneous uptake of BCECF (44). Cells were excited at 490 and 440 nm, and the emission was measured at 530 nm using a Videoscope KS-1381 intensifier tube coupled to a Dage 72 charge-coupled device camera. Images were digitized, stored to a hard disk, and analyzed at a later time using commercially available equipment (Image 1/FL; Universal Imaging, West Chester, PA). Those cells at the lateral tubule wall with BCECF uptake (identified as fluorescence at the isosbestic point, 440 nm) clearly greater than the adjacent cells on either side were identified as intercalated cells. Any cell not classified as an intercalated cell was classified as a principal cell (44). Using cells only at the lateral wall of the tubule minimizes fluorescence from out-of-focus cells, and we have previously shown that this technique allows separate assessment of pHi regulation in the two OMCDis cell populations (44). The fluorescence ratio of each cell was measured and converted to pHi using calibration with the high-K+-nigericin technique (41, 42, 44) at the end of the experiment. Rates of pHi recovery were measured using least-squares linear regression during the initial period after acid loading while the pHi change was linear.
Acid loading. Cells were acid loaded using the ammonia prepulse technique (31). Ammonium chloride, 40 mM, was added to the peritubular solution, using equimolar substitution for sodium chloride, for 5 min, then removed, resulting in abrupt intracellular acidification. Because basolateral Na+/H+ exchange is the primary mechanism of OMCDis principal cell pHi regulation (31, 44), we inhibited this transporter in these studies; the resultant Na+/H+ exchange-independent pHi regulation is a measure of proton secretion. We added ethylisopropylamiloride (EIPA), 1 µM, to the peritubular solution at the beginning of the ammonia prepulse and continued it for ~10 min after ammonia removal. We then removed peritubular EIPA to allow pHi recovery to baseline. Chemicals. Molecular Probes (Eugene, OR) supplied BCECF-AM, which we stored frozen as a 30 mM stock solution in DMSO. Research Biochemical supplied EIPA, which we stored as a 1 mM solution in DMSO. Sch-28080, i.e., 3-cyanomethyl-2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine, was the kind gift of Dr. James Kaminski at Schering (Bloomfield, NJ). All other chemicals were from Sigma Chemical (St. Louis, MO). Statistics. Results are presented as means ± SE; n is reported as the number of cells studied in the number of OMCDis. We analyzed data using unpaired Students t-test and ANOVA, as appropriate, and used P < 0.05 as evidence of statistical significance. We calculated buffer capacity ( ![]() |
RESULTS |
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Proton secretion in the absence of proton pump
inhibitors. The
OMCDis principal cell extrudes
protons via basolateral
Na+/H+
exchange-dependent and -independent mechanisms (44). Because Na+/H+
exchange is the predominant mechanism of principal cell acute pHi regulation (44), but does not
contribute to apical proton secretion, we inhibited it by adding EIPA
(1 µM), a potent amiloride analog (20), to the peritubular solution.
Figure 1 shows a representative experiment,
and Fig. 2 and Table
1 summarize the results. Following an acute
intracellular acid load, principal cell EIPA-insensitive pHi recovery averaged 0.224 ± 0.032 pH units/min (P < 0.001 vs. zero, n = 28 cells in 6 OMCDis). The presence of
EIPA-insensitive proton secretion in the principal cell is consistent
with our previous report (44) and confirms that the principal cell
secretes protons.
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Role of
H+-K+-ATPase
in principal cell pHi
regulation.
One major family of proton transporters expressed in the kidney is
H+-K+-ATPase.
Because mRNA for the -subunit of
H+-K+-ATPase
is expressed in the OMCDis
principal cell (11), we hypothesized that a potassium-dependent proton
transporter might be expressed in this cell. To test this hypothesis,
we examined the extent to which luminal potassium removal altered
pHi regulation. These experiments
are summarized in Fig. 2 and Table 1. In the absence of luminal
potassium, EIPA-insensitive pHi
recovery averaged 0.115 ± 0.023 pH units/min
(n = 15 cells in 3 OMCDis), which was significantly less than in the presence of luminal potassium
(P < 0.002, respectively, by ANOVA).
The OMCDis principal cell appears
to express a luminal potassium-dependent transporter, i.e., an apical
H+-K+-ATPase.
Role of H+-ATPase in principal cell pHi regulation. Because an apical H+-K+-ATPase mediates only 50% of principal cell EIPA-insensitive pHi regulation, a second family of proton transporters may be present. Since a major family of potassium-independent proton transporters expressed in the kidney is the vacuolar-type H+-ATPase (15), we hypothesized that H+-ATPase might contribute to OMCDis principal cell pHi regulation. To test this possibility, we used bafilomycin A1, which is a relatively specific H+-ATPase inhibitor at the concentrations used, 5 nM (45). Figure 2 and Table 1 summarize these results. Luminal bafilomycin A1 addition, 5 nM, decreased potassium-independent pHi recovery to 0.037 ± 0.006 pH units/min (n = 22 cells in 5 OMCDis), significantly less than in the absence of bafilomycin A1 (P < 0.05 by ANOVA). The observation of luminal potassium-independent, luminal bafilomycin A1-sensitive proton secretion suggests that an apical H+-ATPase contributes to OMCDis principal cell proton secretion.
Intercalated cell
pHi regulation.
The OMCDis intercalated cell also
exhibits EIPA-insensitive proton secretion, which differs in its rate
and regulation from the principal cell (44). Yet no studies have
specifically examined the mechanism of
OMCDis intercalated cell proton
secretion. The next set of studies was designed to determine the
mechanism of intercalated cell proton secretion. A representative
experiment showing pHi regulation
in the OMCDis intercalated cell is
shown in Fig. 3, and Fig.
4 and Table 2
summarize the results. Intercalated cell
pHi recovery from an intracellular
acid load averaged 0.434 ± 0.058 pH units/min
(n = 41 cells in 6 OMCDis). Furthermore, as shown
in Fig. 5,
OMCDis intercalated cell proton
secretion, measured as the EIPA-insensitive
pHi recovery rate, is greater than
in the principal cell, similar to our previous findings (44).
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DISCUSSION |
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These studies are the first to examine functionally the mechanisms of apical proton secretion by the OMCDis principal cell and intercalated cell. An apical H+-K+-ATPase mediates ~50% of principal cell pHi regulation. An apical H+-ATPase is also present, which mediates ~50% of principal cell proton secretion. The intercalated cell secretes protons at approximately twice the rate of the principal cell and uses transport mechanisms that are pharmacologically similar. These observations have important implications for our understanding of the cells involved in potassium reabsorption and the transporters involved in potassium reabsorption and proton secretion.
Several previous studies showed that one or more H+-K+-ATPase isoforms play an important role in OMCDis acid-base transport. In some studies, a Sch-28080-sensitive H+-K+-ATPase mediated 40-60% of transepithelial bicarbonate reabsorption (4, 38). In other studies that examined the entire OMCDis and did not separately examine the two OMCDis cell populations, an apical H+-K+-ATPase mediated either ~50% of Na+/H+ exchange-independent pHi regulation (23) or did not contribute to Na+/H+ exchange-independent pHi regulation (18). The reason why an apical H+-K+-ATPase did not contribute to OMCDis pHi regulation in one report (18) is not clear. The current findings are consistent with those previous studies showing H+-K+-ATPase activity in the OMCDis and, more importantly, extends these studies by showing that apical H+-K+-ATPase activity is present in both the principal cell and the intercalated cell.
The finding that the principal cell expresses an apical H+-K+-ATPase suggests that a physiological role of the OMCDis principal cell may be luminal potassium reabsorption. This possibility is supported by the observations that potassium deficiency increases OMCDis potassium reabsorption (46) and induces hypertrophy of OMCDis principal cells along with an increase in OMCDis principal cell apical and basolateral membrane length (13, 17, 50), which suggests that cellular transport is activated. The current study is the first to demonstrate a mechanism for potassium reabsorption by OMCDis principal cell, namely, an apical H+-K+-ATPase. In contrast, there is little evidence that the OMCDis principal cell secretes potassium. First, under basal conditions, the OMCDis does not secrete potassium (46). Second, the OMCDis principal cell apical membrane has a very low conductance, suggesting that apical potassium channels are either not present or are inactive (21). Finally, potassium loading does not cause hypertrophy of the OMCDis principal cell (16, 34). The OMCDis principal cell thus contrasts with the CCD principal cell, which secretes potassium, has a significant apical conductance, and undergoes hypertrophy in response to potassium loading but not potassium restriction (29, 34).
When viewed within the context of the known distribution and
characteristics of renal
H+-K+-ATPase
isoforms, our results suggest that a novel
H+-K+-ATPase
-isoform may be expressed in the
OMCDis principal cell. At least
two families of
H+-K+-ATPase
-subunits have been identified (47, 49).
HK
1 was the first
H+-K+-ATPase
identified in the kidney (48), but neither
HK
1 protein nor mRNA has been
identified in the OMCDis principal
cell under basal conditions (1, 5, 48). A second family of
H+-K+-ATPase
-isoforms, the "colonic
H+-K+-ATPase"
family, consists of HK
2a (49)
and its alternative splice transcripts
HK
2b and
HK
2c (2, 9, 10).
HK
2a mRNA is not expressed in
the OMCDis principal cell under
normal conditions (2), suggesting it does not mediate proton secretion
in this cell. HK
2b is Sch-28080
insensitive (22), suggesting that it does not mediate the
Sch-28080-sensitive proton secretion identified in the present study.
Moreover, the probe used by Ahn et al. (2) to examine the in situ
localization of HK
2a would be
expected to cross-hybridize with
HK
2b. The absence of probe
localization to the OMCDis
principal cell in their studies further suggests that
HK
2b is not the HK
subunit
mediating proton secretion. The third member of the
HK
2 subunit family is
HK
2c (9, 10). Neither the
cellular location nor the pharmacological sensitivity of
HK
2c has been reported, making
it difficult to determine whether HK
2c is expressed in the
OMCDis principal cell. However,
preliminary results in our laboratory suggest this isoform is present
in more than just the intercalated cells of the
OMCDis. Taken as a whole, these
considerations suggest that an
H+-K+-ATPase
-isoform other than HK
1,
HK
2a, and
HK
2b may be expressed in the
OMCDis principal cell.
In addition to reabsorbing potassium, the
OMCDis principal cell may also
secrete protons and reabsorb luminal bicarbonate. This cell possesses
apical H+-ATPase, cytoplasmic
carbonic anhydrase (30), and basolateral Cl/HCO
3
exchange activity (44), in addition to apical
H+-K+-ATPase
activity. Thus it expresses several transporters and enzymes characteristic of an acid-secreting collecting duct cell. Although immunoreactive H+-ATPase is not
present in the OMCDis principal
cell (3, 5, 33, 37, 39), rod-shaped particles, an ultrastructural
hallmark of H+-ATPase, are present
(30, 35). Why immunohistochemical studies do not detect
H+-ATPase in the
OMCDis principal cell is not
clear. One possibility is that an immunologically distinct
H+-ATPase isoform may be present.
Alternatively, the level of
H+-ATPase expression may be below
levels of detection using immunohistochemistry. At present we cannot
differentiate between these possibilities.
In contrast to potassium reabsorption, proton secretion may not be a major role of the OMCDis principal cell. Structure-function studies show that respiratory and metabolic acidosis do not cause morphological changes associated with increased transport, such as hypertrophy, changes in the surface density of the apical plasma membrane, or changes in the tubulovesicular structures in the apical plasma region of the cell (17, 25, 26).
The intercalated cell secretes protons using transport mechanisms, at
least pharmacologically, similar to those used by the principal cell.
The presence of potassium-dependent, Sch-28080-sensitive pHi regulation is consistent with
the presence of HK1
immunoreactivity and mRNA in the
OMCDis intercalated cell,
suggesting that HK
1 may be
expressed and may contribute to intercalated cell proton secretion (1,
5, 48). Alternatively, an
H+-K+-ATPase
-isoform similar to that present in the principal cell may be
present. The presence of luminal potassium-independent, luminal
bafilomycin A1-sensitive
pHi regulation suggests the
presence of an apical H+-ATPase
and is consistent with numerous studies showing an apical H+-ATPase in the
OMCDis intercalated cell (3, 6, 8,
33, 37, 39).
Although the OMCDis principal cell
and intercalated cell utilize pharmacologically similar transport
mechanisms, multiple lines of evidence demonstrate that the principal
cell and intercalated cell are not a single cell population in
different states of functional activity. Immunohistochemical studies
using intercalated cell markers, such as antibodies to
HK1,
H+-ATPase, and band 3 protein,
clearly identify separate intercalated cell and principal cell
populations in both the rat and rabbit (3, 5, 6, 8, 32, 33, 37, 39,
48). Similarly, principal cell markers, such as aquaporin-2,
aquaporin-3, and basolateral
Na+-K+-ATPase,
identify a specific principal cell population (12, 14, 19, 30).
Transmission electron microscopy, scanning electron microscopy, and
freeze-fracture microscopy identify two distinct cell populations with
characteristics of intercalated cells and principal cells that do not
interconvert in response to physiological stimuli (13, 17, 24, 35).
More important, the OMCDis
principal cell and intercalated cell respond differently to
physiological stimuli. For example, potassium restriction causes hypertrophy of both the principal cell and intercalated cell, whereas
acute respiratory acidosis and acute and chronic metabolic acidosis
cause hypertrophy of only the intercalated cell (17, 25). Our studies
show that mineralocorticoids have differing effects on the
OMCDis intercalated cell and
principal cell (44). Although the ultrastructural differences between
the intercalated cell and principal cell may not be as great in the
rabbit as in the rat (30), clear differences exist. A subpopulation of
cells has increased numbers of mitochondria and subapical vesicles
compared with the other population (30). By immunohistochemistry, one population expresses basolateral band 3 protein reactivity and apical
H+-ATPase, typical characteristics
of an intercalated cell, whereas a second population of cells does not,
which is characteristic of a principal cell (32, 33). In contrast, some
studies have identified only a single
OMCDis cell population. Some of
these studies used peritubular loading of BCECF (7, 18, 23); however,
this technique loads intercalated cells and principal cells similarly
(40) and cannot be used to identify separate principal cell and
intercalated cell populations. Another study showed that all
OMCDis cells have similar
electrophysiological characteristics, namely, the presence of a large
apical fractional resistance (21). Our proposed model for the
OMCDis principal cell suggests
that it reabsorbs potassium via an apical
H+-K+-ATPase
and that it does not reabsorb sodium via apical cation channels, which
is consistent with the presence of a large apical fractional resistance
(21). In aggregate, two cell populations appear to be
present in the OMCDis, a principal
cell population and an intercalated cell population.
The unique features of the OMCDis principal cell suggest that it should be distinguished from the principal cell of the CCD. The OMCDis principal cell differs sufficiently from the CCD principal cell by ultrastructural criteria that some authors have suggested it should be termed an "inner stripe cell" (30). The data in the current study also show that the principal cells of the OMCDis should be distinguished from the principal cells of the CCD. However, we suggest that referring to this cell as an OMCDis principal cell both emphasizes its numerous differences from the OMCDis intercalated cell and differentiates it from the CCD principal cell while maintaining consistency with an extensive literature that differentiates the cortical and outer medullary collecting ducts into two cell populations, an intercalated cell and a principal cell population. More important than the name attached to this cell is the recognition that its physiological role and regulation differs from the CCD principal cell.
The observation that both the principal cell and intercalated cell secrete protons and reabsorb luminal bicarbonate raises the question as to the relative importance of the two cells to net proton secretion. Proton secretion rates can be estimated from the rate of EIPA-insensitive pHi recovery, cell volume, cell buffer capacity, and cell number. Intercalated cell Na+/H+ exchange-independent pHi rate is approximately twice that of the principal cell (Ref. 44 and the current study), intercalated cell size is either similar to (13) or slightly larger than principal cell size (35), and intercalated cell buffer capacity is either similar to (44) or slightly greater (current study) than principal cell buffer capacity. At the same time, principal cells are several-fold more abundant than intercalated cells (24, 27). Taking each of these factors into consideration, we suggest that total principal cell proton secretion is similar to total intercalated cell proton secretion under basal conditions. Moreover, the presence of proton secretion by both cell populations in the OMCDis may explain the generally greater rates of transepithelial bicarbonate reabsorption than in the CCD or the outer stripe of the outer medullary collecting duct, where principal cells do not secrete protons (23, 41).
Simultaneous proton secretion by H+-ATPase and H+-K+-ATPase raises the question as to the relative importance of these two families of proteins in OMCDis luminal acidification. In the current study an apical H+-K+-ATPase mediated ~50% of both principal cell and intercalated cell proton secretion. This is similar to the 35-60% seen in studies examining transepithelial bicarbonate reabsorption (4, 28, 38). Our results also show that an apical H+-ATPase mediates the ~50% of proton secretion that does not occur via H+-K+-ATPase. This, too, is consistent with the studies that show an H+-ATPase mediates ~40-65% of proton secretion (4, 38) and show that inhibition of both H+-K+-ATPase and H+-ATPase essentially completely inhibits proton secretion (4, 38).
In summary, the current study identifies several new findings regarding
proton and potassium transport by
OMCDis cells. First, the principal
cell has an apical
H+-K+-ATPase,
suggesting that this cell may play an important role in active
potassium reabsorption. Second, the principal cell
H+-K+-ATPase
-subunit may be a unique
-subunit, differing from
HK
1, HK
2a, and
HK
2b. Third, the principal cell
possesses an apical H+-ATPase
which, in conjunction with the previously identified cytoplasmic carbonic anhydrase and basolateral
Cl
/HCO
3
exchange activity, suggests that the principal cell contributes to
urinary acidification and luminal bicarbonate reabsorption. Finally,
OMCDis intercalated cell proton
transporters are functionally similar to those expressed by the
principal cell, suggesting the possibility that an
H+-K+-ATPase
-subunit similar to that expressed in the principal cell may
contribute, under some circumstances, to intercalated cell proton
secretion. Further studies are needed to identify the specific H+-K+-ATPase
isoforms expressed in the OMCDis
principal cell and intercalated cell, as well as the specific
regulation of the different proton transporters in the
principal cell and intercalated cell.
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ACKNOWLEDGEMENTS |
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We thank April R. Starker for technical assistance and Gina Cowsert for secretarial support.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-45788 (to I. D. Weiner) and DK-49750 (to C. S. Wingo) and by Department of Veterans Affairs Merit Review Grants to I. D. Weiner and to C. S. Wingo.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
1
In this report, we shall use the
H+-K+-ATPase
nomenclature suggested by Wingo et al. (49). We shall identify the
H+-K+-ATPase
-subunit originally described in the gastric parietal cell as
HK
1. The colonic isoform of the
H+-K+-ATPase
-subunit has been previously identified as
HK
2 (49), but recent reports
identify probable alternatively spliced transcripts in the rat,
HK
2b (22), and in the rabbit,
HK
2c (10). Because of these
additional HK
2-related
isoforms, we shall refer to the isoform of
H+-K+-ATPase
originally isolated from the colon as
HK
2a.
Address for reprint requests and other correspondence: I. D. Weiner, Division of Nephrology, Hypertension and Transplantation, Univ. of Florida College of Medicine, P. O. Box 100224, Gainesville, FL 32610-0224 (E-mail: WeineID{at}ufl.edu).
Received 16 September 1998; accepted in final form 23 December 1998.
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