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CELLULAR |
1 Department of pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK
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Abstract |
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(Received 17 July 2006;
accepted after revision 15 August 2006;
first published online 17 August 2006)
Corresponding author S. B. Hladky: Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1PD, UK. Email: sbh1{at}cam.ac.uk
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Introduction |
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The transporters discussed in this paper are found in many cells throughout the body where they are involved in pH regulation and secretion. There have been several studies of pH regulation in vascular endothelia that do not secrete (see, e.g. Faber et al. 1998; Sun et al. 1999) and a great many on HCO3– transport in secretory epithelia and a variety of cell lines (see, e.g. Reinertsen et al. 1988; Tonnessen et al. 1990; Romero et al. 2004 and references therein). Nevertheless because these tissues either subserve different functions or have different embryological origins the transport activities in brain microvascular endothelial cells cannot be inferred from studies on any of these systems.
Because the area of the blood brain barrier is so large, approximately 100 cm2 g–1 (see, e.g. Bradbury, 1979), the rate of secretion of fluid and bicarbonate across the blood–brain barrier expressed per unit area is small. Partly for this reason there is at present no in vitro system that will allow direct, quantitative measurement of the net rate of HCO3– secretion across the endothelial cells. However, the transporters responsible for the secretion will per force also influence the intracellular pH, pHi, of the endothelial cells. Transporters implicated in regulation of pHi in various cell types include the sodium–hydrogen exchangers and several members of the bicarbonate transporter superfamily. The Na+/H+ exchangers, the NHEs (gene family SLC9), extrude protons in exchange for extracellular Na+ (Wakabayashi et al. 1997; Putney et al. 2002). They either are active under most circumstances (e.g. NHE3 in kidney proximal tubules) or more commonly are activated by a fall in pHi. Members of the NHE family can be inhibited by amiloride and more specifically by its derivative, ethylisopropylamiloride (EIPA; Vigne et al. 1983; Kleyman & Cragoe, 1988). The bicarbonate transporter superfamily (SLC4; Romero et al. 2004) contains Na+-independent Cl–/HCO3– exchangers, also termed anion exchangers (AE1, AE2, and AE3; Alper et al. 2001), Na+-driven Cl–/HCO3– exchangers (NDCBE; Boron, 2001; Grichtchenko et al. 2001), and Na+–HCO3– cotransporters (NBC; Romero & Boron, 1999; Soleimani & Burnham, 2001). It is currently unclear if NCBE is a Na+-driven Cl–/HCO3– exchanger or a Na+–HCO3– cotransporter (Wang et al.; Romero et al. 2004). AE activity is increased at alkaline pHi (Reinertsen et al. 1988; Alper et al. 2001) which leads to HCO3– efflux from the cell; NBC, NDCBE or NCBE activity normally leads to HCO3– influx (with the important exception of the 1Na+–3HCO3– mode of activity of NBCe1 in kidney proximal tubules). Members of the multifunctional anion exchanger family (SLC26) of transporters (Mount & Romero, 2004) can also transport HCO3– and OH–. With the exception in some tissues but not others of the electroneutral Na+–HCO3– cotransporter, NBCn1 (SC4A7; Choi et al. 2000; Praetorius et al. 2004a; Bouzinova et al. 2005; Boedtkjer et al. 2006; Damkier et al. 2006), all of the known isoforms of the HCO3–-dependent transporters can be inhibited by the stilbene derivative 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) (Mount & Romero, 2004; Romero et al. 2004).
With an internal pH perhaps 0.3 less than that of the extracellular fluid and a membrane potential at least as negative as –30 mV, H+ will passively enter and HCO3– will passively leave rat brain endothelial cells via any channels or other available routes. The cells must therefore possess some means of extruding acid or equivalently of acquiring base. In cells such as brain endothelial cells, which are not called upon to generate large pH gradients, the normal source of energy for the acid extrusion is the Na+ concentration gradient generated by the Na+ pump. Brain endothelial cells must also mediate a net flux of HCO3– from blood to brain. We present evidence here for three transport activities seen at intracellular pH near the presumed resting value as indicated in Fig. 1 along with ‘channel-like’ permeability and the Na+ pump. Throughout this paper we follow the usual convention (see, e.g. Bevensee & Boron, 1998b) and refer to acid loaders and acid extruders even though much of the acid loading occurs via efflux of HCO3– and much of the acid extrusion occurs via influx of HCO3–.
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Methods |
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Unless otherwise stated, all materials were obtained from Sigma (Poole, Dorset, UK). The compositions of the various buffered solutions are indicated in Table 1.
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The acetoxymethyl ester of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) (Molecular Probes, Netherlands) at 0.5 mM, EIPA at 100 mM, DIDS at 125 mM and ethoxzolamide at 100 mM were prepared in anhydrous DMSO (41648, Fluka, Buchs, Germany). All except DIDS were aliquoted and frozen; DIDS solutions were prepared on the day of the experiment. Nigericin was stored as a 10 mM stock in ethanol. The final concentrations of DMSO or ethanol never exceeded 0.2%.
Animals
Wistar rats (200–350 g, Charles River, Ramsgate, UK) were killed using carbon dioxide asphyxiation in accordance with the Animals (Scientific Procedures) Act 1986.
Cell preparation
Brain microvessels were isolated from the cortical grey matter removed from rats using a method previously described (Abbott et al. 1992; Barrand et al. 1995) and were maintained as described by Seetharaman et al. (1998). Two to three days prior to experiments, cells were plated onto a small area of a glass coverslip coated first with poly D-lysine and then collagen. Medium was replaced 1–2 days before the experiment, and cells were used at passages 2–4.
Measurement of intracellular pH
Measurement of pHi was performed using the pH-sensitive fluorescent dye, BCECF, and a fluorescence spectrophotometer (F-2000, Hitachi). The cuvette was maintained at 37°C and the solutions were stirred by a small magnetic flea throughout the experiments. Fluorescence was recorded at 5 s intervals at 526 nm with excitation alternately at 440 nm and 502 nm using 5 nm (or in some experiments 10 nm) excitation and emission slit widths.
Approximately (2–3) x 104 cells were plated and grown on glass coverslips in a region slightly larger than the 2 mm x 7 mm rectangle illuminated by the excitation beam. For experiments with Hepes-buffered medium, the coverslip with adherent cells was rinsed and mounted in the cuvette containing 2.5 ml of experimental solution without BCECF-AM. After recording the ‘cells-only’ backgrounds at the two excitation wavelengths, the cuvette was exchanged for another containing medium and 0.5 µM BCECF-AM (0.1% DMSO) and the initial medium fluorescence was noted. The coverslip holder, coverslip and cells were then transferred to the new cuvette. The fluorescence at both wavelengths increased linearly with time during loading. At the end of loading, typically for 900 s, the coverslip holder was transferred to a fresh cuvette and the final fluorescence of the loading medium noted. Changes in experimental solutions were made by transfer of the coverslip between cuvettes.
The procedures for experiments with CO2/HCO3–-buffered medium included minor modifications to reduce loss of CO2. Solution equilibrated with 5% CO2–95% O2 by bubbling were taken up into a syringe and dispensed into the cuvette through a short length of wide bore tubing. Care was taken not to produce bubbles or an aerosol. The cuvette was then closed off with a snug-fitting Teflon cap. The lack of change of pHi during three successive transfers of cells between cuvettes is shown in a figure reported previously (Hladky et al. 2000) demonstrating that the layer of buffered solution adherent to the coverslip provided adequate protection for the cells during the transfer. Since there were only a small number of cells in the cuvette during an experiment and the buffer volume was 2.5 ml, the amount of CO2 generated from cellular metabolism was too small to produce any detectable changes in pHi. The ease with which CO2/HCO3–-buffered solutions can be handled in these experiments is a major advantage of the cuvette-based technique.
Calculation of cell associated fluorescence ratios
The initial, ‘cells-only’ readings were taken for use as the background fluorescence values (i.e. those not resulting from cell associated BCECF). pHi was calculated as described below from the ratio (measured fluorescence, excitation 502 nm – background, excitation 502 nm)/(measured fluorescence, excitation 440 nm – background, excitation 440 nm). To confirm the validity of using the ‘cells only’ readings for the background corrections, pHi was also calculated from the ratio of the slope of the cell-associated fluorescence versus time curve measured at 502 nm during loading to that at 440 nm. Cell-associated fluorescence during loading was in turn calculated as the difference between the fluorescence measured in the presence of the cells and the fluorescence of the medium, which were each shown to increase linearly with time during loading. In the few experiments for which there were discrepancies between the values of pHi determined just before the end of loading or just after loading, the data were discarded. All pHi values reported in this paper are calculated from raw fluorescence values that are at least twice (usually more than three times) the background (at both excitation wavelengths).
Calibration of BCECF and the fluorimeter
The pH calibration curve used to convert background-corrected fluorescence ratios to values of pH depends on the properties of the fluorescent indicator, the light source and the detection system. An initial calibration curve was constructed using values of pHi imposed with the nigericin high-K+ method (Thomas et al. 1979; Bevensee & Boron, 1998a) (see Fig. 2A) using 140 mM K+ and 20 mM Cl– and fitted using the standard equation for titration between the acidic and basic forms of the indicator:
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0.2 pH units). This effect of altering Cl– outside on the fluorescence of BCECF inside suggests that intracellular ion concentrations are changed during the calibration procedure, which may invalidate the assumption made that pHi = pHo.
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It was confirmed that in free solution the fluorescence ratios were not affected by Cl–, NH4+, or EIPA over the ranges of concentration encountered in this study. DIDS in solution produced fluorescence somewhat lower than the typical background values. As only changes in pH in the continuous presence or absence of DIDS are interpreted in this paper and the pH calibration curve is approximately linear over the entire region of interest, no correction was made. It was also confirmed that the fluorescence ratios were independent of dye concentration over a range producing emission intensities spanning that recorded in the experiments with cells. The ratio differed by less than 2% even for emission intensities 5 x higher. There has been a previous report (Hegyi et al. 2004) demonstrating that the calibration curve for BCECF and a microscope based optical system can vary with fluorescence intensity. The experiments reported here differ from those in that a fluorimeter has been used instead of a microscope, the detectors are different, and the dye concentrations are lower.
Calculation of intracellular buffering
The non-bicarbonate buffering in the endothelial cells was estimated from the immediate change in pHi following withdrawal of CO2/HCO3– or after the addition or removal of NH3/NH4+. It was not useful to make stepwise changes because pHi changed too rapidly after each step. The intrinsic buffering was calculated as:
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This value was determined as 6.11 ± 0.01 (n = 6) under the conditions used to prepare the experimental solutions by measuring the pH produced by additions of 135 mM NaHCO3–135 mM NaCl at 37°C with constant bubbling with 5% CO2–95% O2.
The data (see Fig. 2C) were fitted using the integrated, single-buffer expression for the ratio of the base added to the change in pHi:
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Following a 200 s period in one dye free test solution, cells were exposed to a second test solution for 200 s. As indicated in Table 3 paired comparisons are either between these two periods, or between the second periods for coverslips from the same preparation measured on the same day.
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Following a 200 s period in dye-free solution, cells were exposed to 20 mM NH4Cl (replacement of Na+ by NH4+). The presence of the NH4Cl results in a rapid initial intracellular alkalinization followed by a slow acidification. After 200 s the cells were transferred to a NH4Cl-free Hepes-buffered solution and the subsequent changes in pHi measured. In all cases the cells undergo a marked intracellular acidification followed by a slower alkalinization (Boron & De Weer, 1976; Roos & Boron, 1981; Thomas, 1984). The recovery process was fitted as a single exponential decay towards a constant value using the solver in Excel.
RT-PCR analysis
Total RNA was isolated from freshly dissected rat brain and cultured rat brain endothelial cells using TriPureTM Isolation Reagent (Roche Diagnostics, Burgess Hill, W. Sussex, UK) according to manufacturer's instructions. All samples were checked for integrity and relative content of RNA by electrophoretic separation through agarose and volumes adjusted accordingly before proceeding to RT-PCR analysis. cDNA synthesis was performed with Bioscript (Bioline Ltd, London) according to the manufacturer's instructions. Samples were stored at –20°C. PCR was performed with a Rotor-Gene 3000TM (Corbett Research, Concord, NSW, AUS) using SensiMix DNA Kit (Quantace Ltd, Watford, UK) (12.5 µl enzyme mix, 0.5 µl Sybr green I stock, 0.75 µl 50 mM MgCl2) together with 500 nM primers and 8.75 µl template in 25 µl final volume. Initial denaturation for 10 min at 95°C was followed by 45 cycles of amplification (95°C for 15 s and 60°C for 60 s) and melting curve analysis between 60 and 99°C in 1°C, 5 s steps.
Primer sequences are listed in Table 2. The results were analysed with the Rotor-Gene software v6 (Corbett Research, Concord, NSW, AUS) using the comparative quantification feature to compare expression of the mRNA of interest with that of NHE1. Sizes of the PCR products were confirmed using agarose gel separation.
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Data are expressed as mean values ± S.E.M. with n indicating the number of experiments. P values are calculated using a paired or unpaired (unequal variance) two-tailed Student's t test as appropriate. The parameters of the buffer curve and the offset of the pH calibration curve were fitted by ordinary least squares minimization. The significance of the improvement in fit allowed by introducing a variable parameter was assessed by an F test on the reduction in the residual sum of squares compared to the mean squared error per remaining degree of freedom (the variance ratio test). Analysis of variance for 429 measurements for cells in Hepes which were derived from 13 primary cultures showed that the variation of measurements between cells derived from a single primary culture (root mean squared deviation 0.09 pH units) was significantly less than the variation between the mean values for each culture (root mean squared deviation 0.2 pH units; P < 10–6). Thus the ranges of values reported in this paper reflect primarily variations in the properties of the cells rather than inaccuracy in the measurements. To minimize the effect of this variation, most measurements are paired with controls measured on the same day and, when possible, on the same cells (see Tables 3 and 4).
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Results |
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For cells loaded with the indicator BCECF in solutions buffered either with Hepes alone or with Hepes plus CO2/HCO3–, the pHi after loading decreased gradually at a rate that remained uniform for more than 400 s. The value immediately after loading, called here the initial pHi, was 7.098 ± 0.004 (n = 473) for cells in Hepes, which is close to but significantly less than the equivalent value for cells loaded in the presence of CO2/HCO3–, 7.146 ± 0.007 (n = 115, P < 0.001).
Because the rate of decrease of pHi was less in the presence of HCO3– than in its (nominal) absence ((1.8 ± 0.2) x 10–4 s–1 and (2.8 ± 0.3) x 10–4 s–1, n = 34, P < 0.02, paired), the difference between the values of pHi for cells in Hepes- or HCO3–-buffered solutions increased during the experiments. Thus it appears that cells are better able to maintain pHi in HCO3–-buffered solution. To test this suggestion further, experiments were performed in which the solutions were interchanged. Following a change from HCO3– + Hepes to Hepes (see Fig. 3), pHi rapidly increased; this corresponds to an efflux of CO2 from the cells and conversion of intracellular HCO3– and H+ to water and CO2. Subsequently, pHi decreased and after 400 s was always lower than before the change (
= 0.14 ± 0.02, P = 0.04, 7 out of 7). Similarly following a change from Hepes to Hepes + HCO3– solutions, pHi initially decreased but after 300 s was higher (in 6 out of 7 experiments) than before the change ( = 0.07 ± 0.02, n = 7, P = 0.04).
Rapid change in pHi when CO2 is added or withdrawn requires carbonic anhydrase within the cells to catalyse the interconversion of CO2 and HCO3–. It may also be aided by carbonic anhydrase exposed to the stirred external solution as there is evidence that a carbonic anhydrase isoform (CAIV) is present on the external surface of these cells (Ghandour et al. 1992). The importance of carbonic anhydrase was confirmed by the observation that the membrane-permeant carbonic anhydrase inhibitor, ethoxzolamide (6-ethoxy-2-benzothiazolesulphonamide) (Maren, 1963; Cousin et al. 1975) clearly blunts the response (see Fig. 3). The increase in pHi measured 20 s after CO2/HCO3– removal was reduced from 0.63 ± 0.6 to 0.17 ± 0.4 by 2 µM ethoxzolamide and to 0.18 ± 0.18 by 20 µM (n = 5, P < 0.002 at both concentrations). Similar effects were observed with acetazolamide (data not shown).
Rate of cellular acid gain and loss in the presence or absence of HCO3–
The resting state pHi of these cells (strictly the slow baseline rate of acid gain for cells in the experimental solutions) is presumed to be governed by a balance between the rates of acid loading and acid extrusion (see Discussion). As shown above, the rate of change in pHi was smaller for cells in the presence than in the absence of HCO3–. The cells are, however, more strongly buffered in the CO2/HCO3–-buffered solutions (at pHi 7.1 ßtotal = 48 mM in the presence of HCO3– compared to 25 mM in its nominal absence) and thus the rate of gain of acid calculated from the rate of change in pHi was nearly the same with the two buffer solutions (see Table 3 rows 1 and 2 and Table 4 row A). This implies that the extra mechanisms of transport brought into play by the presence of HCO3– produce cancelling effects with little overall change in the rate of acid loading.
For cells that are not required to generate large differences in pH, the commonly encountered means for acid extrusion, which are indicated in Fig. 1, entail either exchange of intracellular H+ for external Na+ or cotransport of HCO3– and Na+ into the cell. The most commonly encountered means for acid loading are Cl–/HCO3– exchange and channel-like permeability to H+ or HCO3–. Therefore experiments involving removal of Na+ or Cl– were undertaken in the absence or presence of HCO3– (see Tables 3 and 4) as described below.
Evidence for the presence of a Na+/H+ exchanger
Experiments to demonstrate the presence of a NHE-like Na+/H+ exchanger were conducted in the nominal absence of HCO3–. Replacement of Na+ with NMDG+ (Table 3 row 3) (see Fig. 4) and addition of 5 µM EIPA (Table 3 row 4) produced equal, small increases (Table 4 row C) in the rate of acid gain. Because EIPA is a selective inhibitor of the NHE transporters this suggests that a transporter of the NHE family is the major mechanism of Na+-dependent acid extrusion in the nominal absence of HCO3–. The extrusion partially offsets a somewhat larger rate of acid loading.
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0.5 µM (least squares fit including the data for NMDG+ as an estimate of the non-inhibitable recovery). DIDS at 250 µM, which should not inhibit NHE- mediated transport, did not produce any significant inhibition (P = 0.38 paired, n = 8).
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In the presence of HCO3–, inhibition (or reversal) of Na+-dependent transport by replacement of Na+ with NMDG+ produced a larger increase in the rate of acid gain (Table 3 row 5) (see Fig. 4) than was seen in the absence of HCO3– (Table 3 row 3) suggesting the presence of a Na+- and HCO3–-dependent acid extruder. Removal of Na+ after prior removal of Cl– (see below) still produced a clear increase in the rate of acid gain (Table 3 row 10), implying that most, possibly all, of the Na+-dependent HCO3– transport is Cl– independent. DIDS is an inhibitor of many forms of Cl– and HCO3– transport including acid loaders (such as the AEs) and acid extruders (such as the NBCs). Addition of 250 µM DIDS blocked the increase in rate of acid gain seen when Na+ was removed (compare Table 3 row 6 with row 5). These are the properties expected if Na+-dependent HCO3– transport into the cells is mediated by a transporter like NBCe1, NBCe2 or NBCn1 (Romero et al. 2004; Boedtkjer et al. 2006; Damkier et al. 2006).
Evidence for the presence of an AE-like Na+-independent Cl–/HCO3– exchange
In the absence of Na+, a higher rate of acid gain was seen in the presence of HCO3– than in its absence (Table 4 row D). This implies the presence of a Na+-independent, HCO3–-dependent acid loader. Removal of external Cl– in the presence of HCO3– (see Fig. 7A) produced an alkaline shift in pHi (Table 3 row 7) followed by a return to a net rate of acid gain (Table 3 row 8). This can be interpreted as a transient reversal of a Cl–/HCO3– exchanger followed by its inactivity when internal Cl– is depleted. Removal of Cl– in the absence of Na+ (Table 3 row 9) produced a change in the rate of acid gain similar to that seen in the presence of Na+ (Table 3 row 7).
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Evidence for the expression of HCO3– transporters in brain microvascular endothelial cells
Expression at the mRNA level of NBCe1, NBCe2, NBCn1, NCBE, AE1, AE2 and AE3 calculated relative to that for NHE1 was investigated using real-time PCR (see Fig. 8). A product with a well-defined melting point and of the expected size was found with primers specific for each of these transporters. Transcripts of NHE1, AE2 and NBCn1 were comparably abundant with that for NBCe1 present at ca 1/3rd this level, while those for AE1, AE3, NBCe2, NCBE and NDCBE were detected but at lower levels. For comparison data are also reported for mRNA isolated from chloroid plexus (isolated as in Redzic et al. 2005) and whole kidney, which was used as a positive control. While the relative levels of expression of mRNA do not imply the same relative levels of expression for protein, these results do provide suggested molecular candidates for the transporter functions, i.e. AE2 for Cl–/HCO3– exchange and either NBCn1 or NBCe1 for the Na+–HCO3– cotransporter.
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Discussion |
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Rat brain endothelial cells maintained in primary culture and then minimally disturbed were found to have a basal pHi estimated to be 7.15 ± 0.01. This value is close to the values observed in previous studies, 7.18 ± 0.02 (Hsu et al. 1996) and 6.9 ± 0.08 (Sipos et al. 2005). The slow acidification of these cells under our experimental conditions suggests that some effect of transfer of the cells from growth medium to the experimental solutions, loading with BCECF and illumination has either reduced an acid extrusion system or increased an acid-loading system (which may be a channel-like permeability) to disturb the balance which must obtain in the growing cells. This slow acidification, like the normal balance, is the net result of competing processes. The results presented here provide evidence for activity in rat brain endothelial cells of several different ion transporters (see Fig. 1). These include a Na+/H+ exchanger, a DIDS-sensitive Na+–HCO3– cotransporter that continues to function in the absence of Cl– and a DIDS-sensitive Na+-independent Cl––HCO3– exchanger. This study reports the first evidence in brain microvascular endothelial cells for Na+-independent Cl–/HCO3– exchange and Cl–-independent Na+-HCO3– cotransport and the first determinations of the rates of Na+/H+ exchange and Na+–HCO3– cotransport for internal pH near the resting value. The results also provide the first demonstration of carbonic anhydrase activity in these cells. The transport rates observed near pH 7.1 are modest, which may explain why they have not been reported previously. But, nevertheless, they are sufficiently large that if the transporters are appropriately disposed on the two sides of the cells they could account for the net secretion of HCO3– across the blood–brain barrier.
In the absence of Na+, the presence of CO2/HCO3– increases the net rate of acid loading in the cells, implying the existence of a Na+-independent HCO3– exit mechanism. This mechanism is inhibited by DIDS. In the presence of HCO3–, removal of external Cl– produces a transient alkalinization as if it were the reversal of a Cl–/HCO3– exchanger. In this explanation the reversal is transient because it is limited by the supply of Cl– within the cells.
In the presence of HCO3– removal of Na+ produces a sustained increase in the rate of acid gain which in many experiments was preceded by a short period of more rapid acid gain (see Fig. 4). The behaviour is plausibly explained as transient reversal of the Na+-dependent acid extrusion process followed by its inactivity. The sustained rate of acid gain would then result from the combination of the ‘channel-like’ permeability and the continued function of the Cl–/HCO3– exchanger. Indeed the activity of the exchanger may be increased, as the abolition of Cl– entry via the Na+–K+–2Cl– cotransporter, NKCC1 (O'Donnell et al. 2004; Foroutan & O'Donnell, 2005), will lead to decreased intracellular Cl– concentration and hence a more favourable Cl– gradient for Cl–/HCO3– exchange.
The transient net acid extrusion when Cl– is removed in the presence of HCO3– (see Fig. 7A) is simply and plausibly explained as reversal of Cl–/HCO3– exchange. However, if the only effect of Cl– removal is on the anion exchanger, the rate of the sustained acid gain after the transient should be slower than the rate observed in the presence of Cl–. This has not been observed (Table 3, row 9). This is evidence that Cl– removal has an additional effect. The data could be explained if Cl– removal also inhibits a component of acid extrusion like that which would be mediated by a Na+ driven Cl–/HCO3– exchanger, e.g. NDCBE or possibly NCBE. The present data are consistent with this type of explanation but without quantitative modelling taking into account factors like changes in membrane potential they cannot be said to require it. Comparison of the increase in the rate of acid gain produced by Na+ removal in the absence of Cl– (Table 3 row 10) with that seen in the presence of Cl– (Table 3 row 5) suggests that the major component of Na+-coupled HCO3– transport is Cl– independent.
The properties of the acid extrusion and the acid loading in the presence of HCO3– correspond to those that could be mediated by known transporters. Na+ and HCO3– dependence, DIDS sensitivity and independence of Cl– are properties of many NBC-like Na–HCO3– cotransporters (including NBCe1, NBCe2 and, possibly, NBCn1) which would function as an acid extruder in these cells. The mRNAs for NBCe1 and NBCn1 are clearly present in the cells. Cl– and HCO3– dependence, Na+ independence and DIDS sensitivity are properties of the Cl–/HCO3– exchangers of the AE family and some members of the anion exchanger (SLC26) superfamily (Mount & Romero, 2004). There is clear expression of mRNA for AE2 in these cells.
In the nominal absence of HCO3– the principal acid extruder appears to be an NHE because the mechanism is inhibited by Na+ removal or by EIPA, a selective inhibitor of the NHEs, and it becomes much more prominent as pHi is reduced. In addition to mRNA for NHE1 these cells also express mRNA for NHE2, 3 and 4 (Sipos et al. 2005) and NHE5 (Taylor et al. 2002). In the absence of Na+ there is still a significant rate of recovery from an acid load imposed by an NH4Cl prepulse that reduces pHi to 6.3. Sipos et al. (2005) reported no recovery in the absence of Na+, but the acid loads they imposed reduced pHi only to 6.5. At the lower pHi of 6.3 the electrochemical gradient for H+ is likely to be outwards and that for HCO3– inwards and the recovery could be mediated by a ‘channel-like’ permeability or reversal of the Cl–/HCO3– exchanger.
Molecular candidates for the transporters mediating the functions observed can be suggested by the pattern of expression levels of mRNA observed using real-time PCR. A comparison between mRNA derived from brain microvascular endothelial cells, choroid plexus epithelial cells and extracts from whole kidney (used as a positive control) is shown in Fig. 8. For each tissue or cell type the expression levels have been normalized to the expression for NHE1 as that transporter is expected to be highly expressed in all. The patterns of expression are very different. The striking feature of the results for the kidney is the prominent expression of NBCe1, which is hardly surprising as basolateral NBCe1 and apical NHE3 in proximal tubule cells mediate the bulk of renal HCO3– reabsorption. In choroid plexus there is high expression of NCBE, NBCn1 and NBCe2. This is consistent with the results from PCR and immunohistochemistry reported previously (Praetorius et al. 2004b; Bouzinova et al. 2005; Damkier et al. 2006). There is also clear expression of NHE1 consistent with previous binding studies (Kalaria et al. 1998). However, it is should be noted that NHE proteins have not been detected in choroid plexus using immunohistochemistry (Praetorius et al. 2004b). In the brain endothelial cells the prevalent mRNA species are those for the reference Na+/H+ exchanger NHE1, the Cl–-independent Na+–HCO3– cotransporters, NBCn1 and NBCe1 and the Na+-independent Cl–/HCO3– exchanger, AE2. mRNAs for NCBE, which may be a Na+-dependent Cl–/HCO3– exchanger, NDBCE, AE1, AE3 and NBCe2 were detected but at substantially lower abundance. Conventional PCR studies (amplification followed by visualization on gels) using RNA isolated from brain microvascular endothelial cells have previously shown expression for NHE1 and 5 (Taylor et al. 2002) and NHE1, 2, 3 and 4 (Sipos et al. 2005).
Table 5 presents a comparison of the transport activites observed in vascular endothelia from blood vessels outside the central nervous system. These have in common with brain microvascular endothelial cells the need to regulate intracellular pH but differ in that they do not mediate a net secretion of fluid. Estimates of the rates of transport exist only for endothelial cells from human umbilical vein. In that tissue the dominant mode of HCO3– entry at resting pH is Cl– dependent while in the brain microvascular cells it is Cl– independent. Damkier et al. (2006) have recently shown that NBCn1 is present in endothelial cells from many locations in the vasculature outside the CNS, which suggests an important role for Cl–-independent Na+–HCO3– cotransport in peripheral endothelia as well.
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25 µM s–1 x 0.5 µm = 1.25 pmol cm–2 s–1. This flux is small compared to those seen in transporting epithelia but nevertheless large enough to account for current estimates of HCO3– secretion across the blood–brain barrier. Assuming that the surface area of the capillaries in brain tissue is 100 cm2 g–1 (see, e.g. Bradbury, 1979) and for a 1000 g brain, this secretion rate is 10 mmol day–1. Thus the transporters described here could account for current estimates, 1–5 mmol day–1, of net secretion of HCO3– into the brain (Milhorat, 1987; Cserr & Patlak, 1992; Abbott, 2004; Redzic & Segal, 2004). In summary when cultured rat brain endothelial cells are transferred from growth medium to a HCO3–-buffered solution, the modest requirement for acid efflux appears to be largely but not completely met by Na+–HCO3– cotransport. Following intracellular acidification, as might occur in vivo during hypoxia, both Na+/H+ exchange and Na+–HCO3– cotransport (with or without exchange for Cl–) (Sipos et al. 2005) is likely to be important in the responses of the cells. HCO3– secretion by the endothelial cells requires a means of base entry into the cells (or equivalently acid extrusion) on one surface of the cell and of base extrusion (acid loading) on the other. This study provides evidence for at least two mechanisms for acid extrusion, an NBC-like Na+–HCO3– cotransporter and an NHE-like Na+/H+ exchanger, and two mechanisms for acid loading, channel-like permeability and an exchanger. The most important acid extruder in HCO3–-buffered solutions is a Na+–HCO3– cotransporter (possibly NBCn1 or NBCe1). The transporters identified functionally in vitro can account for secretion of HCO3– provided the Na+-linked influx of HCO3– occurs primarily across the luminal membrane while the Na+-independent efflux of HCO3– occurs primarily across the abluminal surface. Multiple molecular candidates for the transporters that may fulfil these roles have been identified from real-time PCR measurements of mRNA. Determining which are present at the level of protein and how they are localized within the cells will be the subject of future studies.
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Abbott NJ, Hughes CCW, Revest PA & Greenwood J (1992). Development and characterisation of a rat brain capillary endothelial culture: Towards an in vitro blood–brain barrier. J Cell Sci 103, 23–37.
Alper SL (1994). The band 3-related AE anion-exchanger gene family. Cellular Physiol Biochem 4, 265–281.[CrossRef]
Alper SL, Chernova MN & Stewart AK (2001). Regulation of Na+-independent Cl–/HCO3– exchangers by pH. JOP 2 (Suppl), 171–175.[CrossRef][Medline]
Aronson PS (1985). Kinetic-properties of the plasma-membrane Na+-H+ exchanger. Annu Rev Physiol 47, 545–560.[CrossRef][Medline]
Barrand MA, Robertson KJ & von Weikersthal SF (1995). Comparisons of P-glycoprotein expression in isolated rat brain microvessels and in primary cultures of endothelial cells derived from microvasculature of rat brain, epididymal fat pad and from aorta. FEBS Lett 374, 179–183.[CrossRef][Medline]
Betz AL (1983). Sodium transport in capillaries isolated from rat brain. J Neurochem 41, 1150–1157.[CrossRef][Medline]
Betz AL, Firth JA & Goldstein GW (1980). Polarity of the blood–brain barrier: distribution of enzymes between the luminal and antiluminal membranes of brain capillary endothelial cells HCO3–. Brain Res 192, 17–28.[CrossRef][Medline]
Bevensee MO & Boron WF (1998a). Fluorescence indicators. In pH and Brain Function, ed. Kaila K & Ransom BR, pp. 129–151. Wiley-Liss, New York.
Bevensee MO & Boron WF (1998b). pH regulation in mammalian neurons. In pH and Brain Function, ed. Kaila K & Ransom BR, pp. 211–231. Wiley-Liss, New York.
Boedtkjer E, Praetorius J & Aalkjaer C (2006). NBCn1 (slc4a7) mediates the Na+-dependent bicarbonate transport important for regulation of intracellular pH in mouse vascular smooth muscle cells. Circ Res 98, 515–523.
Bonanno JA (2003). Identity and regulation of ion transport mechanisms in the corneal endothelium. Prog Retinal Eye Res 22, 69–94.[CrossRef][Medline]
Bonanno JA & Giasson C (1992a). Intracellular pH regulation in fresh and cultured bovine corneal endothelium. 2. Na+-HCO3–cotransport and Cl–/HCO3– exchange. Invest Ophthalmol Visual Sci 33, 3068–3079.
Bonanno JA & Giasson C (1992b). Intracellular pH regulation in fresh and cultured bovine corneal endothelium. 1. Na+/H+ exchange in the absence and presence of HCO3–. Invest Ophthalmol Visual Sci 33, 3058–3067.
Boron WF (2001). Sodium-coupled bicarbonate transporters. JOP 2 (Suppl), 176–181.[Medline]
Boron WF & De Weer P (1976). Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors. J General Physiol 67, 91–112.
Bouzinova EV, Praetorius J, Virkki LV, Nielsen S, Boron WF & Aalkjaer C (2005). Na+-dependent HCO3– uptake into the rat choroid plexus epithelium is partially DIDS sensitive. Am J Physiol 289, C1448–C1456.[CrossRef]
Boyarsky G, Ganz MB, Sterzel RB & Boron WF (1988). pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO3–. Am J Physiol 255, C844–C856.[Medline]
Bradbury MWB (1979). The Concept of a Blood–Brain Barrier. Wiley, Chichester, UK.
Brown PD, Davies SL, Seake T & Millar ID (2004). Molecular mechanisms of cerebrospinal fluid production. Neuroscience 129, 957–970.[CrossRef][Medline]
Chaillet JR & Boron WF (1985). Intracellular calibration of a pH-sensitive dye in isolated, perfused salamander proximal tubules. J General Physiol 86, 765–794.
Choi I, Aalkjaer C, Boulpaep EL & Boron WF (2000). An electroneutral sodium/bicarbonate cotransporter NBCn1 and associated sodium channel. Nature 405, 571–575.
Choi I, Rosas JD, Kobayashi C & Boron WF (2002). Functional characterization of ‘NCBE’, a Na/HCO3 cotransporter. FASEB J 16, A796.
Cousin JL, Motais R & Sola F (1975). Transmembrane exchange of chloride with bicarbonate ion in mammalian red blood cells: evidence for a sulphonamide-sensitive ‘carrier’. J Phys
, continuous line) using the nigericin–high K+ method or pH in a solution of BCECF (
, dashed line) plotted against the background corrected fluorescence ratio that was produced. The calibration curve used in the rest of this paper is indistinguishable on this graph from the solution curve. B, example of null-point offset data for addition of a 6: 1 ratio of butyric acid to trimethylamine (20 mM total). pH, as calculated using the cells curve in A, is plotted against the change in pH that was observed. The straight line is fitted by linear regression with intercept 7.15 which is 0.14 units higher than the null-point pH, 7.01, for the 6: 1 ratio. C, chord estimates of buffering versus pHi either just after loading cells with base or just before a load of acid or base is removed. The chord estimate is the amount of base added divided by the change in pH. x, addition of NH4Cl; 
, removal of NH4Cl; O, removal of CO2/HCO3–; –, the theoretical predictions for a single intracellular buffer with pK = 6.44 and maximum capacity 74 mM.
