Apical proton secretion by the inner stripe of the outer medullary collecting duct

I. David Weiner, Amy E. Frank
Charles S. Wingo
(With the Technical Assistance of Wulf Sullivan)

Division of Nephrology, Hypertension and Transplantation, University of Florida College of Medicine, Gainesville 32610; and Gainesville Veterans Affairs Medical Center, Gainesville, Florida 32608


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, HKalpha 1 protein1 and mRNA, and HKalpha 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 HKbeta subunit is expressed in the OMCDis principal cell (11), which suggests that this subunit may associate with a novel alpha -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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (beta T) as beta T = Delta [NH+4]i/Delta pHi, where [NH+4]i is the intracellular NH+4 concentration.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Inner stripe of outer medullary collecting duct (OMCDis) principal cell ethylisopropylamiloride (EIPA)-insensitive intracellular pH (pHi) regulation. Example of OMCDis principal cell acid loaded using the ammonia prepulse technique and demonstrating both EIPA-insensitive and -sensitive pHi regulation.


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Fig. 2.   Inhibitor sensitivity of OMCDis principal cell EIPA-insensitive pHi regulation. Rates of EIPA-insensitive pHi regulation are plotted in pH units per minute. Buffer capacity was not significantly different in any of the protocols shown. Sch-28080 concentration was 10 µM. Baf, 5 nM bafilomycin A1. NS, not significant.

                              
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Table 1.   Effect of proton pump inhibitors on principal cell EIPA-insensitive pHi regulation

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 beta -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.

To confirm further the presence of apical H+-K+-ATPase, we examined the effect of the H+-K+-ATPase inhibitor, Sch-28080, on pH regulation. These results are summarized in Fig. 2 and Table 1. In the presence of luminal Sch-28080 (10 µM), EIPA-insensitive pHi recovery from an acid load averaged 0.131 ± 0.028 pH units/min (n = 24 cells in 6 OMCDis). Sch-28080 significantly inhibited principal cell proton secretion (P < 0.003 by ANOVA), and the effect of Sch-28080 did not differ significantly from the effect of luminal potassium removal [not significant (NS) by ANOVA].

We conclude that an apical H+-K+-ATPase, defined functionally as a luminal potassium-dependent, Sch-28080-sensitive proton transporter, is present in the OMCDis principal cell. This transporter mediates ~50% of OMCDis principal cell EIPA-insensitive proton secretion.

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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Fig. 3.   OMCDis intercalated cell EIPA-insensitive pHi regulation: example of OMCDis intercalated cell acid loaded by the ammonia prepulse technique, demonstrating both EIPA-insensitive and -sensitive pHi regulation.


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Fig. 4.   Inhibitor sensitivity of OMCDis intercalated cell EIPA-insensitive pHi regulation. Rates of EIPA-insensitive pHi regulation are plotted in pH units per minute. Buffer capacity was not significantly different in any of the protocols shown. Sch-28080 concentration was 10 µM. Baf refers to bafilomycin A1, 5 nM.

                              
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Table 2.   Effect of proton pump inhibitors on intercalated cell EIPA-insensitive pHi regulation



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Fig. 5.   Comparison of principal cell and intercalated cell EIPA-insensitive pHi recovery: rate of pHi recovery measured in the period of linear pHi recovery following an intracellular acid load induced by the ammonium chloride prepulse technique.

Because HKalpha 1 protein (5, 48) and mRNA (1) and HKalpha 2a mRNA (2) are expressed in the OMCDis intercalated cell, we hypothesized that an H+-K+-ATPase may contribute to intercalated cell H+ secretion. To test this possibility, we examined whether luminal potassium removal would alter OMCDis intercalated cell pHi regulation. These results are summarized in Fig. 4 and Table 2. In the absence of luminal potassium, intercalated cell EIPA-insensitive pHi recovery averaged 0.204 ± 0.037 pH units/min (n = 24 cells in 3 OMCDis), which is significantly less than when luminal potassium was present (P < 0.001 by ANOVA). Thus the OMCDis intercalated cell expresses an apical potassium-dependent proton transporter, most likely an apical H+-K+-ATPase.

To confirm that this transporter is an H+-K+-ATPase, we examined the effect of the H+-K+-ATPase inhibitor, Sch-28080, on pHi regulation. These results are summarized in Fig. 4 and Table 2. In the presence of luminal Sch-28080, 10 µM, OMCDis pHi recovery averaged 0.163 ± 0.032 pH units/min (n = 37 cells in 6 OMCDis). Thus luminal Sch-28080 significantly inhibited intercalated cell pHi recovery (P < 0.001 by ANOVA).

These results show that the OMCDis intercalated cell possesses a luminal potassium-dependent, luminal Sch-28080-sensitive pHi regulatory mechanism, functionally identifying an apical H+-K+-ATPase. There was no significant difference between the effect of luminal potassium removal and luminal Sch-28080 addition (P = NS by ANOVA).

The presence of luminal potassium-independent pHi regulation suggests that proton transporters other than H+-K+-ATPase may be functional in the OMCDis intercalated cell. Because immunoreactive H+-ATPase is present in this cell (5, 48), we next examined whether an H+-ATPase mediated EIPA-insensitive pHi regulation. These results are summarized in Fig. 4 and Table 2. Luminal bafilomycin A1 significantly inhibited potassium-independent pHi recovery, decreasing it to 0.029 ± 0.004 pH units/min (P < 0.005 by ANOVA, n = 30 cells in 5 OMCDis). These results provide evidence for a luminal potassium-independent, bafilomycin A1-sensitive transporter, functionally identifying an apical H+-ATPase, in the OMCDis intercalated cell.

The effects of luminal potassium removal, luminal Sch-28080 addition, and luminal bafilomycin A1 addition on OMCDis EIPA-insensitive pHi regulation could reflect either differences in proton secretion rates or differences in either cellular buffer capacity or the degree of acid loading (31). Buffer capacity averaged 14.7 ± 1.1 meq · l-1 · pH unit-1 in the principal cell and 20.1 ± 1.0 meq · l-1 · pH unit-1 in the intercalated cell, measurements similar to what we have reported previously (44). None of the experimental conditions significantly altered buffer capacity in either the principal cell or the intercalated cell (P = NS by ANOVA). Nor did the inhibitors used alter the degree of acid loading induced by the ammonium chloride prepulse (P = NS by ANOVA). The differences in EIPA-insensitive pHi regulation in the different experimental protocols cannot be ascribed to differences in either the degree of acid loading or cellular buffer capacity.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -isoform may be expressed in the OMCDis principal cell. At least two families of H+-K+-ATPase alpha -subunits have been identified (47, 49). HKalpha 1 was the first H+-K+-ATPase identified in the kidney (48), but neither HKalpha 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 alpha -isoforms, the "colonic H+-K+-ATPase" family, consists of HKalpha 2a (49) and its alternative splice transcripts HKalpha 2b and HKalpha 2c (2, 9, 10). HKalpha 2a mRNA is not expressed in the OMCDis principal cell under normal conditions (2), suggesting it does not mediate proton secretion in this cell. HKalpha 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 HKalpha 2a would be expected to cross-hybridize with HKalpha 2b. The absence of probe localization to the OMCDis principal cell in their studies further suggests that HKalpha 2b is not the HKalpha subunit mediating proton secretion. The third member of the HKalpha 2 subunit family is HKalpha 2c (9, 10). Neither the cellular location nor the pharmacological sensitivity of HKalpha 2c has been reported, making it difficult to determine whether HKalpha 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 alpha -isoform other than HKalpha 1, HKalpha 2a, and HKalpha 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 HKalpha 1 immunoreactivity and mRNA in the OMCDis intercalated cell, suggesting that HKalpha 1 may be expressed and may contribute to intercalated cell proton secretion (1, 5, 48). Alternatively, an H+-K+-ATPase alpha -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 HKalpha 1, 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 alpha -subunit may be a unique alpha -subunit, differing from HKalpha 1, HKalpha 2a, and HKalpha 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 alpha -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.


    ACKNOWLEDGEMENTS

We thank April R. Starker for technical assistance and Gina Cowsert for secretarial support.


    FOOTNOTES

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 alpha -subunit originally described in the gastric parietal cell as HKalpha 1. The colonic isoform of the H+-K+-ATPase alpha -subunit has been previously identified as HKalpha 2 (49), but recent reports identify probable alternatively spliced transcripts in the rat, HKalpha 2b (22), and in the rabbit, HKalpha 2c (10). Because of these additional HKalpha 2-related isoforms, we shall refer to the isoform of H+-K+-ATPase originally isolated from the colon as HKalpha 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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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