1Institute for Physiology and Biophysics, 2Institute of Anatomy, and 3The Water and Salt Research Center, University of Aarhus, Aarhus, Denmark; and 4Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut
Submitted 29 June 2005 ; accepted in final form 9 August 2005
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ABSTRACT |
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bicarbonate metabolism; BCECF; cerebrospinal fluid; acid/base transport; ammonium prepulse
Epithelial fluid secretion depends on the unidirectional transport of ions, creating the osmotic gradient that drives the movement of water. Unidirectional transport of ions can be achieved through a polarized distribution of ion transport proteins in the apical and basolateral membranes of epithelial cells (25). The central component in CSF secretion is the continuous active Na+ transport, which is brought about by Na+-K+-ATPase, creating a substantial inward chemical gradient for Na+. Many other transporters utilize the developed inward Na+ gradient to transport other molecules across cell membranes against their chemical gradients as, e.g., Na+-solute exchangers and cotransporters. In the CP, the Na+-K+-ATPase is localized to the apical plasma membrane domain of the epithelia (21, 24, 29), and is thereby directly involved in the secretion of Na+ ions. In contrast to most other epithelia, both secretory and absorptive, the basolateral plasma membrane becomes the "bottleneck" in the overall transepithelial transport of Na+.
Several observations point to the involvement of stilbene-sensitive basolateral Na+-driven HCO3 uptake in pH regulation of the CP epithelial cells and in CSF production. First, a major component of the Na+ extrusion into the CSF has been shown to be coupled to the secretion of HCO3 (10), and the apical fluid production is sensitive to changes in the basolateral HCO3 concentration in vitro (8). Also, the basolateral HCO3 entry into frog CP was stilbene sensitive and utilized the Na+ gradient as its driving force (22). The pH recovery from intracellular acidification of cultured CP epithelial cells was Na+ and HCO3 dependent and was prevented by the application of a stilbene-type inhibitor (11). Acetazolamide, a carbonic anhydrase inhibitor, reduced CSF secretion by 3080% (27). This inhibitory effect was associated with a decreased Na+ entry into CSF. Thus, identifying the basolateral Na+-dependent HCO3 transport, which possibly contributes to the Na+ entry into CP epithelial cells, becomes of major interest for better understanding CSF production.
The molecular expression and basolateral localization of two electroneutral sodium-bicarbonate cotransporters was shown using RT-PCR and immunohistochemical analysis (16). Both transporters belong to the Slc4a gene family of bicarbonate transporters: the NBCn1 (Slc4a7), which is an electroneutral sodium bicarbonate cotransporter (3), and the Na+-driven Cl bicarbonate exchanger (NCBE) (Slc4a10): a Na+-dependent chloride bicarbonate exchanger (4, 7, 28). It should be noted that the chloride dependence of the latter transporter has been questioned in a meeting abstract (4). Finally, the mRNA encoding a third Na+-dependent HCO3 transporter, the NBCe2 or NBC4 was also detected in the CP (16). The NBCe2 protein has not yet been localized to the epithelial cells. All three transporters could function as basolateral HCO3 entry pathways. Thus we examined the cellular and subcellular localization of NBCe2 and measured the bicarbonate-dependent pHi regulation at steady-state level and after cellular acidification to describe the Na+-dependent HCO3 uptake into CP epithelial cells functionally.
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METHODS |
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An 22-amino acid NH2-terminal peptide (MKVEEKAGVKKLEPTSYRRRHPEC) was synthesized on the basis of the published rat Slc4a5 (NBCe2/NBC4) sequence, National Center for Biotechnology Information accession no. AAS98674. Two rabbits were injected with the peptide, and the resulting antisera were affinity purified with the immunizing peptide coupled to an agarose column (Sulfolink; Pierce, Rockford, IL). An antiserum against a 73-amino acid COOH-terminal fusion protein (AF293337 [GenBank] ) of the human NBCe2 was also applied. A previously validated antibody against NCBE (16) was conjugated to Alexa Fluor 488 according to the manufacturer's protocol (Molecular Probes, Eugene, OR) and applied for double-labeling experiments.
Immunohistochemistry
Halothane-anesthetized animals were fixed by retrograde perfusion via the abdominal aorta with 4% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4, and brain and control tissues were removed. The tissues were dehydrated and embedded in paraffin, and 2-µm-thick sections were cut with a rotary microtome (Leica, Heidelberg, Germany). The sections were dewaxed and rehydrated, and endogenous peroxidase was blocked by 0.5% H2O2 in absolute methanol. To reveal antigens, the sections were boiled in 1 mM Tris, pH 9, supplemented with 0.5 mM EGTA. Nonspecific binding of immunoglobulin was quenched by incubating the sections in 50 mM NH4Cl and blocked in PBS supplemented with 1% bovine serum albumin (BSA), 0.05% saponin, and 0.2% gelatin. The sections were incubated overnight at 4°C with primary anti-NH2-terminal or -COOH-terminal NBCe2 antibodies diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. After being washed, the sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (Dako) diluted in PBS supplemented with BSA and Triton X-100. The peroxidase stain was visualized by 0.05% 3,3'-diaminobenzidine tetrahydrochloride dissolved in PBS with 0.1% H2O2. Mayer's hematoxylin was used for counterstaining, and the sections were dehydrated and mounted in hydrophobic Eukitt mounting medium (O. Kindler, Freiburg, Germany). Microscopy was performed on a Leica DMRE bright-field microscope equipped with a Leica DM300 digital camera. For double-labeling immunofluorescence microscopy, the COOH-terminal NBCe2 antibody exposed sections were incubated with Alexa 546-conjugated goat anti-rabbit secondary antibodies (Molecular Probes) in PBS supplemented with BSA and Triton X-100 and then overnight with Alexa Fluor 488-conjugated anti-NCBE antibody. The sections were then mounted with a coverslip in Glycergel antifade medium (Dako) and inspected on a Leica DMRS confocal microscope with an HCX PlanApo x64 magnification (1.32 numerical aperture) objective. The immunofluorescence images were merged with differential interference contrast images to reveal the relationship between the tissue structures and the fluorescence labeling.
Immunogold Electron Microscopy
Tissue blocks prepared from mouse brain were cryoprotected with 2.3 M sucrose containing 2% paraformaldehyde and rapidly frozen in liquid nitrogen. The samples were freeze substituted by sequential equilibration over 3 days in methanol containing 0.5% uranyl acetate at temperatures raised gradually from 80° to 70°C, rinsed in pure methanol for 24 h while the temperature was increased from 70 to 45°C, and infiltrated with Lowicryl HM20 and methanol 1:1, 2:1, and, finally, pure Lowicryl HM20 before ultraviolet polymerization for 2 days at 45°C and 2 days at 0°C. Immunolabeling was performed on ultrathin Lowicryl HM20 sections. Sections were pretreated with a saturated solution of NaOH in absolute ethanol (23 s), rinsed, and preincubated for 10 min with 0.1% sodium borohydride and 50 mM glycine in 0.05 M Tris, pH 7.4, containing 0.1% Triton X-100. Sections were rinsed and incubated overnight at 4°C with the COOH-teminal NBCe2 antibody diluted in 0.05 M Tris, pH 7.4, containing 0.1% Triton X-100 with 0.2% milk. After being rinsed, sections were incubated for 1 h at room temperature with goat anti-rabbit IgG conjugated to 10-nm colloidal gold particles (GAR.EM10, BioCell Research Laboratories, Cardiff, UK). The sections were stained with uranyl acetate and lead citrate before examination in a Philips Morgagni 268D electron microscope.
In Vitro Preparation for Measurements of Intracellular pH
Adult male Munich-Wistar rats (250300 g) from Taconic Europe (M&B, Lille Skensved, Denmark) had free access to water and pelleted food (Altromin, Lage, Germany). On the day of the experiment, rats were euthanized by CO2 inhalation and their brains were rapidly removed and placed in ice-cold physiological salt solution (CO2/HCO3 solution; see Table 1). The entire CP was removed and divided into several pieces of <1-mm length. This procedure seemed to be necessary for gaining access to the basolateral side of the epithelium because in the uncut tissue NH4Cl application failed induce intracellular alkalinization and the acute acidifying effect of CO2/HCO3 was reduced. Afterward, the tissue was allowed to adhere to Cell-Tak (BD Biosciences, Broendby, Denmark)-coated coverslips, which were then placed in a perfusion chamber. The chamber was mounted on an Olympus CK40 microscope equipped with UApo/240 (x40 magnification, 1.35 numerical aperture) oil-immersion objective. A monochromator was used as light source and the computer-controlled detection system included an IC-200 CCD camera (PTI, Lawrenceville, NJ). The tissue was loaded with 16 µM BCECF-AM (Molecular Probes), diluted in HEPES-buffered solution (pH 7.35, CO2/HCO3-free solution, Table 1) for 15 min at room temperature. Excess dye was washed out by continuous perfusion with HEPES-buffered solution for 15 min at 37°C.
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pHi recovery after acid load. The NH4+ prepulse technique was used to acidify the intracellular environment. After a few minutes of baseline pHi recording in the presence of CO2/HCO3, the external solution was changed to 20 mM NH4Cl for 5 min [or to 10 mM (NH4)2SO4 in Cl-free experiments; Table 1]. Cells were then shifted to a Na+-free solution (Table 1) containing 600 µM amiloride (to block Na+/H+ exchange) for 5 min. Finally, Na+ was reintroduced in the continued presence of amiloride.
Measurement of intrinsic buffer capacity and net base fluxes.
The slopes of the pHi recovery traces (pHi/
t) were determined corresponding to a 1-min period after full exchange of the bath solution. The rate of recovery was converted into values of net base flux (Jnet) by multiplication of the rate of pHi recovery by the total buffering capacity. The total buffering capacity (
t) is the sum of the intrinsic buffering capacity (
i) and CO2/HCO3-dependent buffering capacity (
CO2) (5, 20). To obtain
i values, samples were perfused consecutively in the presence of 1 mM furosemide for 300 s or until pHi was steady with HEPES-buffered solution, containing varying concentrations of NH4Cl (20, 10, 5, 0 mM). [NH4+]i was calculated from the Henderson-Hasselbalch equation, and
i was calculated as
[NH4+]i/
pHi;
CO2 was calculated as 2.3 x [HCO3]i (20), where [HCO3]i = (9.95 x 107)/10pHi at PCO2 = 40 mmHg. Net flux of base equivalents was calculated as Jnet =
t x
pHi/
t. A linear fit of
i data points was used for calculations of base fluxes (Fig. 1B). The intrinsic buffering power of CP epithelial cells was relatively constant in the pHi range of 7.1 to 7.75 and was estimated as 10.23 mM/pH unit at pHi 7.39. Jnet was calculated for nine single cells during each experiment. The mean Jnet values were computed from 410 experiments for each protocol. All animal protocols have been approved, and the license for the use of experimental animals was issued by the Danish Ministry of Justice.
Statistical Analyses
Results are presented as means ± SE. Two-tailed paired or nonpaired Student's t-test was used for data analysis, and P < 0.05 was considered statistically significant.
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RESULTS |
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Immunoblotting using the anti-COOH-terminal NBCe2 antibody revealed immunoreactive proteins of the expected 140-kDa band and a broader
180-kDa band in homogenates of NBCe2-injected Xenopus laevis oocytes (Fig. 2A). These bands were not found in water-injected oocytes. The two bands likely represent unglycosylated (140 kDa) and glycosylated (180 kDa) forms of the NBCe2 protein. The anti-COOH-terminal antibody localized NBCe2 to the apical plasma membrane domain of the CP epithelial cells. Figure 2A, bottom, shows a low-magnification image of the immunoperoxidase-labeled mouse CP using the anti-COOH-terminal antibody (Fig. 2A, left). The staining was completely prevented when the primary antibody had been preabsorbed by the immunizing protein (Fig. 2A, right). Similar results were obtained with an anti-NH2-terminal NBCe2 antibody (not shown). The double-staining fluorescence labeling of the rat CP illustrated in Fig. 2B shows similar localization of NBCe2 (anti-COOH-terminal antibody) to the apical membrane domain as in mouse and demonstrates NCBE staining corresponding to the basolateral domain of the rat CP epithelium. Figure 2C reveals immunogold labeling of the apical microvillar projections from the epithelial cells of mouse CP using the anti-COOH-terminal antibody. No other cellular structures, including the basolateral membranes, stained with the anti-COOH-terminal antibody.
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pHi in HEPES and CO2/HCO3-buffered solutions.
The basic approach to study the action of acid/base transporters is the recording of pHi upon changes in the concentration of the transported solutes in the bath. The first objective was to determine whether HCO3 transporters contribute to steady-state pHi in epithelial cells of rat CP. Figure 2D represents a working model of the activity and directions of ion transport in the CP epithelial cells in the presence of HCO3. The mean steady-state pHi of CP epithelium in a HEPES-buffered solution was 7.03 ± 0.02 (n = 41). The pHi reached a significantly higher steady-state value when CO2/HCO3 was introduced to the external solution (Fig. 3A). The mean steady-state pHi was 7.38 ± 0.02 (n = 41), with the pHi averaging 0.35 ± 0.03 pH units (P < 0.001) and an initial HCO3 influx of 0.139 ± 0.035 mM/s. The results indicate that net HCO3-dependent base loading takes place during introduction of the CO2/HCO3. When CO2/HCO3 was removed, pHi transiently rose and then declined more slowly.
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Dependence of steady state pHi on extracellular Na+. Removal of extracellular Na+ in the presence of CO2/HCO3 acidified the cells by 0.33 ± 0.04 pH units from pHi 7.41 ± 0.04 to 7.05 ± 0.04 (n = 7, P < 0.05) (Fig. 4A) in 20 min. This effect was completely reversed after reintroduction of Na+ into the bath. This indicates that Na+-dependent base loading contributes to the elevated pHi in the presence of HCO3. The addition of 200 µM DIDS simultaneously with Na+ hindered the complete Na+-dependent pHi recovery. The pHi level achieved values similar to those shown in Fig. 3 and was 7.25 ± 0.03 (n = 5, P < 0.05, Fig. 4A). Addition of 200 µM DIDS after Na+ removal led to a small but statistically significant 0.10 ± 0.05 unit increase of pHi from 7.05 ± 0.04 to 7.15 ± 0.05 (n = 5, P < 0.01) (Fig. 4B). The inhibition of any reverse mode of the Na+-dependent HCO3 transport, together with the apparent low initial rate of pHi change on addition of DIDS, suggests that the overall flux of acid and base equivalents is low under these conditions. Finally, the reintroduction of Na+ aftereffect of 200 µM DIDS was observed (Fig. 4C), causing the average steady-state pHi to increase from 7.13 ± 0.01 to 7.27 ± 0.02 (P < 0.01, n = 5). This steady-state pHi was significantly (P < 0.01) lower the initial one of 7.38 ± 0.01, indicating once again the presence of at least two DIDS-sensitive and DIDS-insensitive, Na+-dependent mechanisms mediating the steady-state pHi in the presence of CO2/HCO3. It is important to mention that the level of pHi increase after addition of Na+ in the presence of DIDS was similar to the level of acidification in choroidal cells after DIDS application in the continuous presence of Na+ (Fig. 3B).
Influence of extracellular Cl removal on steady-state pHi.
Cl was removed from the bath solution in the presence of CO2/HCO3, to investigate whether Cl-dependent base extrusion was opposing the base loading at steady-state pHi. The pHi rapidly increased from 7.38 ± 0.07 to 7.61 ± 0.03 in response to external Cl removal (n = 8, P < 0.05) (Fig. 5A) and then slowly declined within 46 min to reach a new steady-state level, 0.05 ± 0.01 pH unit above the initial pHi. The pHi returned to the initial level only after the reintroduction of Cl, demonstrating the involvement of Cl-dependent base extrusion in setting the steady-state pHi. The addition of 200 µM DIDS after the Cl-free steady-state pHi was reached led to a decrease of pHi from 7.43 ± 0.07 to 7.28 ± 0.05 (pHi of 0.15 ± 0.04, n = 5, P < 0.01) (Fig. 5B), i.e., identical to the level after DIDS addition during the baseline pHi recording in the presence of Cl.
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Na+ dependence of the pHi recovery.
An alternative way to investigate acid extrusion or base-loading mechanisms is monitoring the recovery of pHi after an acid load (1, 20). Cells are acutely loaded with acid by the NH4+ prepulse technique and the mechanism of pHi recovery investigated by changing composition of solutes and blockers in the bath. Figure 6A depicts the mean traces of such prepulses, where NH4+ was washed out in Na+-free bicarbonate-buffered solution (CO2/HCO3 trace). The pHi between resting conditions and acid load was 0.83 ± 0.07 (n = 8). Recovery of pHi was not observed in the absence of Na+, reflecting the Na+ dependency of the acid extrusion. In contrast, a significant pHi recovery and a corresponding increase in net base influx was observed when Na+ was reintroduced (0.828 ± 0.116 mM/s, n = 8). Na+-dependent recovery in the absence of HCO3 illustrates the pHi dependency of the net base influx (HCO3 line in Fig. 6A). In the absence of CO2/HCO3, the flux was significantly smaller (0.243 ± 0.058 mM/s at pHi = 6.6, P < 0.01, n = 4). In the nominal absence of HCO3 we observed the Na+-dependent amiloride-insensitive recovery of pHi. The molecular basis for this transport remains unknown. The corresponding net H+ flux was considered insignificant compared with the flux in the presence of CO2/HCO3 and was thus not subtracted from other measures of HCO3-dependent fluxes.
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pHi recovery in Cl-free media. The requirement for Cl of the Na+-dependent HCO3 transport at low pHi was tested by preincubating the tissue for 30 min without this anion (and applying a Cl-free NH4+ prepulse). The tissue incubation for 30 min in the Cl-free media was considered efficient for Cl depletion because t for 36Cl uptake and efflux were 12 and 17 s, respectively (9, 17). As shown on Fig. 6, C and D, Na+-dependent recovery and net base flux did occur in the absence of Cl. Although Cl omission modifies the balance between acid and base loaders, the Na+-dependent pHi recovery did occur in the absence of extracellular Cl.
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DISCUSSION |
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At least three Na+-dependent transport proteins from the Slc4a family are present in the rat CP. First, NBCn1 (Slc4a7 gene product) transports the equivalent of one HCO3 with one Na+, using the transmembrane Na+ gradient as a driving force for the electroneutral transport (3). This protein functions as an acid extruder with a seemingly low DIDS sensitivity (3). Second, NCBE (Slc4a10 gene product) is a DIDS-sensitive, electroneutral, Na+-coupled HCO3 transporter that may cotransport the equivalent of two HCO3 with one Na+ in exchange for Cl (28). Third, in the current study, we demonstrate the apical localization of an electrogenic Na+-HCO3 cotransporter, the NBCe2. The immunohistochemical analysis not only supplements the previous detection of NBCe2 mRNA in the isolated CP (16) but also identified the microvilli of the luminal surface as the sole site of expression within the epithelium. NBCe2 (or NBC4) was originally cloned from human heart by Pushkin and coauthors (18) and was functionally characterized by Sassani et al. (23) and Virkki et al. (26).
Given the apical localization, NBCe2 could contribute to either luminal base influx or efflux depending on the electrochemical gradient, but not to the basolateral Na-HCO3 uptake. Two other Na+-HCO3 transporters are present in the CP (16). These are situated in the basolateral plasma membrane of the epithelial cells and are electroneutral. Therefore, they are most likely to mediate net base influx into CP epithelial cells. To demonstrate the Na+-dependent uptake of HCO3 into the epithelial cells of the CP, small pieces of rat choroid plexuses were prepared and the pH changes were studied by fluorescence microscopy. Bilateral access to the cells was evidenced by pH response to NH4+ and CO2 addition as described in METHODS. Furthermore, we sought to determine which of the transport proteins are functionally detectable by probing for DIDS sensitivity and Cl dependence. We used two approaches to identify the Na+-coupled HCO3 transporters functionally in CP epithelial cells: manipulation of bath composition at steady-state pHi and cellular acidification by ammonium prepulsing. These approaches were chosen rather than primary cell culture because our first priority was the demonstration on NaHCO3 transport in the native tissue. The advantage of culturing the CP cells would be easier discrimination of apical and basolateral processes. However, the use of cultured epithelial cells would require documentation for a satisfactory degree of differentiation over time and the absence of other cell types.
The pHi of the CP epithelial cells increased upon addition of CO2/HCO3. This is indicative of the presence of potent HCO3-dependent base loaders, which are alkalizing the cells by overriding the base extrusion until pHi reach 7.4. Interestingly, we did not observe the initial CO2-induced transient acidification often seen in other systems. This could be due to fast HCO3 influx overriding the influx of CO2, and therefore initial transient acidification may not be seen.
The increase of steady-state pHi apparently occurs by the action of partly DIDS-sensitive, Na+-dependent HCO3 transport because removal of sodium as well as addition of DIDS in the presence of CO2/HCO3 lowered the steady-state pHi. DIDS did not fully inhibit the base uptake, because the steady-state pHi in the presence of DIDS did not reach the level that it did in the HCO3-free conditions. The effect of reintroducing Na+ in the presence of DIDS emphasizes the significance of DIDS-insensitive component of Na+-dependent base loading. This could be explained either by the action of a single type of partially DIDS-sensitive transporter or by the combined action of both DIDS-sensitive and -insensitive transporters. In the former case, NBCn1 would be the sole base loader working around steady-state pHi because it is the only Slc4a family member that has no or little sensitivity toward DIDS. However, in many epithelia, the DIDS sensitivity of NBCn1 has been negligible (1315). In the latter case, NBCn1 would seem to be accompanied by DIDS-sensitive base loaders such as NCBE and/or NBCe2, which are both strongly inhibited by 200 µM of the inhibitor (26, 28).
The presence of a DIDS-insensitive component of the Na+-dependent HCO3 transport was also found at lower pHi. In fact, it seemed that the acidified cells demonstrated even less DIDS sensitivity of the Na+-HCO3-dependent base uptake. This base uptake is most likely mediated by NBCn1 because of the aforementioned apparent DIDS insensitivity in epithelia. Interestingly, the net base influx appeared to become significantly DIDS sensitive at around pHi 7.2. Again, this may reflect the action of one or both of the DIDS-sensitive base loaders, NCBE and NBCe2. Alternatively, a single base loader could display varying DIDS sensitivity within the applied range of pH and hence account for the DIDS-insensitive transport at acidic pH and partly DIDS-sensitive base loading near steady-state pHi. However, such pH dependence of DIDS sensitivity has not been described for any protein of the Slc4a gene family to our knowledge. In 1977, Wright (30) showed that bicarbonate concentration dependently influenced the activity of the Na+ pump in the frog CP. Enhanced basolateral Na+/H+ exchange was thought to induce intracellular Na+ accumulation, which in turn would stimulate the pump. According to our findings, accumulation of intracellular Na+ could just as well be mediated directly by basolateral Na+ and HCO3 cotransport through NCBE and/or NBCn1. The apical NBCe2 could also be involved in the intracellular Na+ accumulation under certain conditions, if Na+ and HCO3 are transported inwardly. It should be noted, however, that our current technique does not allow the discrimination of basolateral and apical processes, because both sides are exposed to the same solution. Therefore, it is not possible to ascribe the DIDS-sensitive base transport to any specific transporter.
There are several ways to explain the transient increase in steady-state pHi due to acute removal of extracellular Cl. First, Cl removal may reverse AE2 and thereby increase pHi, and then depletion of intracellular Cl would lead to the AE2 inhibition. In addition, Cl removal would transiently enhance Na+-dependent Cl/HCO3 exchange, because of the reversal of the Cl gradient. An alternative explanation of the transient pHi increase is that the Cl removal causes cell shrinkage due to the loss of intracellular Cl (and indirectly also K+), which, in turn, may activate one or more base loaders. In addition, this activation would be expected to be transient by nature. However, Cl/HCO3 exchange appears to participate in settings of steady-state pHi in the presence of CO2/HCO3.
The tissue incubation for 30 min in the Cl-free media was considered sufficient for Cl depletion because t for 36Cl uptake and efflux were 12 s and 17 s, respectively (9, 17). The NaHCO3-dependent base import after acidification did not seem to depend on the presence of Cl. This is consistent with the lack of DIDS sensitivity of the pHi recovery at low pHi, because NBCn1 is not a Cl/HCO3 exchanger and is relatively DIDS insensitive. After Cl depletion, steady-state pHi was elevated as observed in the steady-state measurements. Therefore, the minimal pHi obtained after NH4Cl-induced acidification was higher in these experiments despite the similar magnitude of the induced pH decrease. Nevertheless, the net base flux was actually increased rather than decreased in the absence of Cl. This increase likely reflects that the base loaders work unopposed when Cl, and thereby AE2 function, is absent.
In conclusion, the electrogenic Na+-HCO3 cotransporter, NBCe2 or NBC4, was immunolocalized to the luminal plasma membrane of CP epithelial cells. Thereby, a total of three NaHCO3 transporters have been localized in this tissue. The other two, NCBE and NBCn1, were previously found to be situated in the basolateral plasma membrane. Furthermore, base equivalents are taken up by the epithelial cells of the CP by Na+, HCO3-dependent pathways, and, to a much smaller extent, by Na+-dependent HCO3-independent transport. At low pHi, the base is taken up largely by DIDS-insensitive base transport, whereas the base uptake is partially DIDS sensitive near steady-state pHi. The DIDS-insensitive component is likely mediated by NBCn1, and the DIDS-sensitive component may reflect NCBE or NBCe2 action. NCBE and NBCn1 proteins most likely mediate cellular Na+ uptake in the CP epithelia, whereas NBCe2 may facilitate HCO3 and Na+ transport from the cells into the CSF or in the opposite direction. In case of a 1 Na+:3 HCO3 stoichiometry and a membrane potential around 60 mV, the net transport through NBCe2 would likely be directed outward. In this way, NBCe2 could contribute to Na+ and thereby CSF secretion. Future studies providing selective access to the basolateral and apical sides of polarized epithelium are warranted to clarify the relative importance of the transporters in pHi regulation and in production of CSF.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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