Na-coupled bicarbonate transporters of the solute carrier 4 family in the nervous system: function, localization, and relevance to neurologic function.

Many cellular processes including neuronal activity are sensitive to changes in intracellular and/or extracellular pH-both of which are regulated by acid-base transporter activity. HCO(3)(-)-dependent transporters are particularly potent regulators of intracellular pH in neurons and astrocytes, and also contribute to the composition of the cerebrospinal fluid (CSF). The molecular physiology of HCO(3)(-) transporters has advanced considerably over the past ∼14 years as investigators have cloned and characterized the function and localization of many Na-Coupled Bicarbonate Transporters of the solute carrier 4 (Slc4) family (NCBTs). In this review, we provide an updated overview of the function and localization of NCBTs in the nervous system. Multiple NCBTs are expressed in neurons and astrocytes in various brain regions, as well as in epithelial cells of the choroid plexus. Characteristics of human patients with SLC4 gene mutations/deletions and results from recent studies on mice with Slc4 gene disruptions highlight the functional importance of NCBTs in neuronal activity, somatosensory function, and CSF production. Furthermore, energy-deficient states (e.g., hypoxia and ischemia) lead to altered expression and activity of NCBTs. Thus, recent studies expand our understanding of the role of NCBTs in regulating the pH and ionic composition of the nervous system that can modulate neuronal activity.

Localization of electrogenic Na/bicarbonate cotransporter NBCe1 variants in rat brain.

The activity of HCO(3)(-) transporters contributes to the acid-base environment of the nervous system. In the present study, we used in situ hybridization, immunoblotting, immunohistochemistry, and immunogold electron microscopy to localize electrogenic Na/bicarbonate cotransporter NBCe1 splice variants (-A, -B, and -C) in rat brain. The in situ hybridization data are consistent with NBCe1-B and -C, but not -A, being the predominant NBCe1 variants in brain, particularly in the cerebellum, hippocampus, piriform cortex, and olfactory bulb. An antisense probe to the B and C variants strongly labeled granule neurons in the dentate gyrus of the hippocampus, and cells in the granule layer and Purkinje layer (e.g. Bergmann glia) of the cerebellum. Weaker labeling was observed in the pyramidal layer of the hippocampus and in astrocytes throughout the brain. Similar, but weaker labeling was obtained with an antisense probe to the A and B variants. In immunoblot studies, antibodies to the A and B variants (alphaA/B) and C variant (alphaC) labeled approximately 130-kDa proteins in various brain regions. From immunohistochemistry data, both alphaA/B and alphaC exhibited diffuse labeling throughout brain, but alphaA/B labeling was more intracellular and punctate. Based on co-localization studies with antibodies to neuronal or astrocytic markers, alphaA/B labeled neurons in the pyramidal layer and dentate gyrus of the hippocampus, as well as cortex. alphaC labeled glia surrounding neurons (and possibly neurons) in the neuropil of the Purkinje cell layer of the cerebellum, the pyramidal cell layer and dentate gyrus of the hippocampus, and the cortex. According to electron microscopy data from the cerebellum, alphaA/B primarily labeled neurons intracellularly and alphaC labeled astrocytes at the plasma membrane. In summary, the B and C variants are the predominant NBCe1 variants in rat brain and exhibit different localization profiles.

Inhibition of the cardiac electrogenic sodium bicarbonate cotransporter reduces ischemic injury.

OBJECTIVE: Although it is believed that sodium-driven acid-base transport plays a central role in the development of the reperfusion injury that follows cardiac ischemia, research to date has demonstrated only a role for Na(+)/H(+) exchange (NHE). However, Na(+)-driven HCO(-)(3) transport, which is quantitatively as important as NHE in cardiac cells, has not been examined. METHODS AND RESULTS: Here the results show that a neutralizing antibody raised against the human heart electrogenic Na(+)/HCO(3)(-) cotransporter (hhNBC) blocked the recovery of pH after acidic pulse both in HEK-293 cells expressing hhNBC and in rat cardiac myocytes demonstrating the presence of an electrogenic NBC in rat cardiac myocytes similar to hhNBC. Administration of anti-NBC antibody to ischemic-reperfused rat hearts markedly protects systolic and diastolic functions of the heart during reperfusion. Furthermore, using a quantitative real-time RT-PCR (TaqMan) and Western blot analysis we demonstrated that in human cardiomyopathic hearts, mRNA and protein levels of hhNBC increase, whereas mRNA levels of the electroneutral Na(+)/HCO(3)(-) cotransporter (NBCn1) remain unchanged. CONCLUSION: Our data provide evidence that inhibition of hhNBC, whose role in cardiac pathologies could be amplified by overexpression, represents a novel therapeutic approach for ischemic heart disease.

Sodium-hydrogen exchangers and sodium-bicarbonate co-transporters: ontogeny of protein expression in the rat brain.

We used western blotting to examine the developmental profiles (at embryonic day 16 and postnatal days 1, 13, 23, 33 and 105) of protein expression for three sodium-hydrogen exchanger isoforms (1, 2 and 4) and for a sodium-bicarbonate co-transporter in three CNS regions (cortex, cerebellum and brainstem-diencephalon). In microsomal preparations, sodium-hydrogen exchanger isoform 1 and sodium-bicarbonate co-transporter protein expression in the CNS increases gradually from embryonic day 16 (25-40% of the adult level) to postnatal day 105. In contrast, sodium-hydrogen exchanger isoform 2 and 4 expression reaches a maximum (three to 20 times the adult level) at around three to four weeks of age. There is significant regional heterogeneity in the expression of sodium-hydrogen exchanger and sodium-bicarbonate co-transporter proteins in the rat CNS. Sodium-hydrogen exchanger isoform 1 was highly expressed in the brainstem-diencephalon, whereas the sodium-bicarbonate co-transporter was robustly expressed in the cerebellum and brainstem-diencephalon. These data indicate that the expression of sodium-hydrogen exchanger and sodium-bicarbonate co-transporter proteins varies as a function of both development and specific brain region.

Na/HCO3 cotransporters in rat brain: expression in glia, neurons, and choroid plexus.

We studied the expression and distribution of Na/HCO(3) cotransporters in rat brain using polynucleotide probes and polyclonal antibodies derived from the electrogenic rat kidney Na/HCO(3) cotransporter (rkNBC). In whole brain, we observed a single mRNA ( approximately 7.5 kb) by Northern hybridization and a major approximately 130 kDa protein by immunoblotting with a polyclonal antiserum directed against the C terminus of rkNBC. NBC mRNA and protein were present in cortex, brainstem-diencephalon, and cerebellum. In situ hybridization revealed NBC mRNA expression throughout the CNS, with particularly high levels in olfactory bulb, hippocampal dentate gyrus, and cerebellum. NBC mRNA was present in glial cells (e.g., Bergmann glia of cerebellum and hippocampal astrocytes) and neurons (e.g., granule cells of dentate gyrus and neurons of cortex or striatum). Double hybridization of mRNA encoding NBC and glutamate transporter 1 (glial marker) confirmed that both glia and neurons express NBC. Indirect immunofluorescence microscopy demonstrated NBC protein throughout the CNS, particularly in hippocampus and cerebellum. Although NBC mRNA was restricted to cell bodies, NBC protein was distributed diffusely, compatible with a localization in cell processes and perhaps cell bodies. Double labeling with glial fibrillary acidic protein (astrocytic marker), microtubule-associated protein 2 (neuronal marker), or 2',3'-cyclic mononucleotide 3'-phosphodiesterase (oligodendrocytic marker) demonstrated expression of NBC protein in specific subpopulations of both glia and neurons. Moreover, NBC protein was present in both cultured hippocampal astrocytes and cortical neurons. NBC mRNA and protein were also present in epithelial cells of choroid plexus, ependyma, and meninges. Our results are thus consistent with multiple novel roles for Na/HCO(3) cotransport in CNS physiology.

An electrogenic Na(+)-HCO(-)(3) cotransporter (NBC) with a novel COOH-terminus, cloned from rat brain.

We screened rat brain cDNA libraries and used 5' rapid amplification of cDNA ends to clone two electrogenic Na(+)-HCO(-)(3) cotransporter (NBC) isoforms from rat brain (rb1NBC and rb2NBC). At the amino acid level, one clone (rb1NBC) is 96% identical to human pancreas NBC. The other clone (rb2NBC) is identical to rb1NBC except for 61 unique COOH-terminal amino acids, the result of a 97-bp deletion near the 3' end of the open-reading frame. Using RT-PCR, we confirmed that mRNA from rat brain contains this 97-bp deletion. Furthermore, we generated rabbit polyclonal antibodies that distinguish between the unique COOH-termini of rb1NBC (alpharb1NBC) and rb2NBC (alpharb2NBC). alpharb1NBC labels an approximately 130-kDa protein predominantly from kidney, and alpharb2NBC labels an approximately 130-kDa protein predominantly from brain. alpharb2NBC labels a protein that is more highly expressed in cortical neurons than astrocytes cultured from rat brain; alpharb1NBC exhibits the opposite pattern. In expression studies, applying 1.5% CO(2)/10 mM HCO(-)(3) to Xenopus oocytes injected with rb2NBC cRNA causes 1) pH(i) to recover from the initial CO(2)-induced acidification and 2) the cell to hyperpolarize. Subsequently, removing external Na(+) reverses the pH(i) increase and elicits a rapid depolarization. In the presence of 450 microM DIDS, removing external Na(+) has no effect on pH(i) and elicits a small hyperpolarization. The rate of the pH(i) decrease elicited by removing Na(+) is insensitive to removing external Cl(-). Thus rb2NBC is a DIDS-sensitive, electrogenic NBC that is predominantly expressed in brain of at least rat.

Effect of trace levels of nigericin on intracellular pH and acid-base transport in rat renal mesangial cells.

Nigericin is an ionophore commonly used at the end of experiments to calibrate intracellularly trapped pH-sensitive dyes. In the present study, we explore the possibility that residual nigericin from dye calibration in one experiment might interfere with intracellular pH (pHi) changes in the next. Using the pH-sensitive fluorescent dye 2', 7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF), we measured pHi in cultured rat renal mesangial cells. Nigericin contamination caused: (i) an increase in acid loading during the pHi decrease elicited by removing extracellular Na+, (ii) an increase in acid extrusion during the pHi increase caused by elevating extracellular [K+], and (iii) an acid shift in the pHi dependence of the background intracellular acid loading unmasked by inhibiting Na-H exchange with ethylisopropylamiloride (EIPA). However, contamination had no effect on the pHi dependence of Na-H exchange, computed by adding the pHi dependencies of total acid extrusion and background acid loading. Nigericin contamination can be conveniently minimized by using a separate line to deliver nigericin to the cells, and by briefly washing the tubing with ethanol and water after each experiment.

Shrinkage-induced activation of Na+/H+ exchange in rat renal mesangial cells.

Using the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), we examined the effect of hyperosmolar solutions, which presumably caused cell shrinkage, on intracellular pH (pHi) regulation in mesangial cells (single cells or populations) cultured from the rat kidney. The calibration of BCECF is identical in shrunken and unshrunken mesangial cells if the extracellular K+ concentration ([K+]) is adjusted to match the predicted intracellular [K+]. For pHi values between approximately 6.7 and approximately 7.4, the intrinsic buffering power in shrunken cells (600 mosmol/kgH2O) is threefold larger than in unshrunken cells (approximately 300 mosmol/kgH2O). In the nominal absence of CO2/HCO-3, exposing cell populations to a HEPES-buffered solution supplemented with approximately 300 mM mannitol (600 mosmol/kgH2O) causes steady-state pHi to increase by approximately 0.4. The pHi increase is due to activation of Na+/H+ exchange because, in single cells, it is blocked in the absence of external Na+ or in the presence of 50 microM ethylisopropylamiloride (EIPA). Preincubating cells in a Cl--free solution for at least 14 min inhibits the shrinkage-induced pHi increase by 80%. We calculated the pHi dependence of the Na+/H+ exchange rate in cell populations under normosmolar and hyperosmolar conditions by summing 1) the pHi dependence of the total acid-extrusion rate and 2) the pHi dependence of the EIPA-insensitive acid-loading rate. Shrinkage alkali shifts the pHi dependence of Na+/H+ exchange by approximately 0.7 pH units.

Intracellular pH regulation in cultured astrocytes from rat hippocampus. I. Role Of HCO3-.

We studied the regulation of intracellular pH (pH) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pH. Intrinsic buffering power (beta) was 10.5 mM (pH unit) at pH 7.0, and decreased linearly with pH; the best-fit line to the data had a slope of -10.0 mM (pH unit). In the absence of HCO, pH recovery from an acid load was mediated predominantly by a Na-H exchanger because the recovery was inhibited 88% by amiloride and 79% by ethylisopropylamiloride (EIPA) at pH 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pH. Acid extrusion was inhibited 68% (pH 6.23) by substituting Li for Na in the bath solution. Switching from a CO/HCO-free to a CO/HCO-containing bath solution caused mean steady state pH to increase from 6.82 to 6.90, due to a Na-driven HCO transporter. The HCO-induced pH increase was unaffected by amiloride, but was inhibited 75% (pH 6.85) by 400 microM 4, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and 65% (pH 6.55-6.75) by pretreating astrocytes for up to approximately 6.3 h with 400 microM 4-acetamide-4'-isothiocyanatostilbene-2, 2'-disulfonic acid (SITS). The CO/HCO-induced pH increase was blocked when external Na was replaced with -methyl--glucammonium (NMDG). In the presence of HCO, the Na-driven HCO transporter contributed to the pH recovery from an acid load. For example, HCO shifted the plot of acid-extrusion rate vs. pH by 0.15-0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO increased acid extrusion 3.8-fold (pH 6.20). When astrocytes were acid loaded in amiloride, with Li as the major cation, HCO failed to elicit a substantial increase in pH. Thus, Li does not appear to substitute well for Na on the HCO transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na-driven HCO transporter are the predominant acid extruders in astrocytes.

Intracellular pH regulation in cultured astrocytes from rat hippocampus. II. Electrogenic Na/HCO3 cotransport.

In the preceding paper (Bevensee, M.O., R.A. Weed, and W.F. Boron. 1997. 110: 453-465.), we showed that a Na-driven influx of HCO causes the increase in intracellular pH (pH) observed when astrocytes cultured from rat hippocampus are exposed to 5% CO/17 mM HCO. In the present study, we used the pH-sensitive fluorescent indicator 2',7'-biscarboxyethyl-5,6-carboxyfluorescein (BCECF) and the perforated patch-clamp technique to determine whether this transporter is a Na-driven Cl-HCO exchanger, an electrogenic Na/HCO cotransporter, or an electroneutral Na/HCO cotransporter. To determine if the transporter is a Na-driven Cl-HCO exchanger, we depleted the cells of intracellular Cl by incubating them in a Cl-free solution for an average of approximately 11 min. We verified the depletion with the Cl-sensitive dye -(6-methoxyquinolyl)acetoethyl ester (MQAE). In Cl-depleted cells, the pH still increases after one or more exposures to CO/HCO. Furthermore, the pH decrease elicited by external Na removal does not require external Cl. Therefore, the transporter cannot be a Na-driven Cl-HCO exchanger. To determine if the transporter is an electrogenic Na/ HCO cotransporter, we measured pH and plasma membrane voltage (V) while removing external Na, in the presence/absence of CO/HCO and in the presence/absence of 400 microM 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS). The CO/HCO solutions contained 20% CO and 68 mM HCO, pH 7.3, to maximize the HCO flux. In pH experiments, removing external Na in the presence of CO/HCO elicited an equivalent HCO efflux of 281 microM s. The HCO influx elicited by returning external Na was inhibited 63% by DIDS, so that the predicted DIDS-sensitive V change was 3.3 mV. Indeed, we found that removing external Na elicited a DIDS-sensitive depolarization that was 2.6 mV larger in the presence than in the absence of CO/ HCO. Thus, the Na/HCO cotransporter is electrogenic. Because a cotransporter with a Na:HCO stoichiometry of 1:3 or higher would predict a net HCO efflux, rather than the required influx, we conclude that rat hippocampal astrocytes have an electrogenic Na/HCO cotransporter with a stoichiometry of 1:2.

Functional expression of the cDNA encoded by the human ATP1AL1 gene.

The human ATP1AL1 gene encodes a protein expressed in brain, kidney, and skin and that is highly homologous to the recently cloned nongastric isoforms of H-K-adenosinetriphosphatase H-K-ATPase). We have generated polyclonal antibodies against the protein encoded by ATP1AL1 and used them to monitor the protein's expression and distribution in transfection studies. The protein was retained in the endplasmic reticulum when it was transiently expressed alone in COS cells. In COS cells cotransfected with ATP1AL1 plus gastric H-K-ATPase beta-subunit cDNAs (ATP1AL1-gH-K beta), both proteins reached the surface. Stably transfected lines of HEK 293 cells expressing both of these proteins demonstrate a 86Rb+ uptake activity sensitive to both 2-methyl,8-(phenylmeoxy)imidazo(1,2-a)pyridine 3-acetonitrile (SCH-28080) and ouabain (inhibitory constants of approximately 131 and 42 microM, respectively). Outward proton fluxes were measured in the same cells as the spontaneous intracellular pH (pHi) recovery in Cells loaded with a pH-sensitive dye [2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein] and subjected to acid loading through an NH4Cl pulse. The cells expressing both the ATP1AL1-encoded protein and the gastric H-K-ATPase beta-subunit possess a net acid extrusion activity that can be inhibited by 1 mM ouabain. Comparison of the 86Rb+ influx and proton efflux, however, does not support equal H+/Rb+ exchange mediated by this pump under the conditions of pHi-monitoring experiments. Moreover, whereas the acid extrusion activity mediated by the pump shows a marked pH dependence, the 86Rb+ uptake activity present in the cells expressing the ATP1AL1-gH-K beta complex cannot be stimulated by acute lowering of pHi. These data suggest that the ATP1AL1-encoded protein is the catalytic alpha-subunit of a human K(+)-dependent ATPase. The possible implications of the discrepancy between 86Rb+ uptake and pHi monitoring data are discussed.

pH regulation in single CA1 neurons acutely isolated from the hippocampi of immature and mature rats.

1. We used the pH-sensitive fluorescent dye 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) to study the regulation of intracellular pH (pHi) in single pyramidal neurons freshly isolated from the hippocampal CA1 region of immature (2- to 10-day-old) and more mature (21- to 30-day-old) rats. 2. Whether isolated from immature or mature rats, neurons had a broad range of initial pHi values (6.3-7.7) when the cells were examined in solutions buffered with Hepes and no CO2/HCO3-. The initial pHi distribution for neurons isolated from immature rats was best fitted with a Gaussian distribution with a mean of 6.95. In contrast, the initial pHi distribution for neurons isolated from mature rats was best fitted with the sum of two Gaussian distributions with means of 6.68 and 7.32. 3. When neurons with a relatively low initial pHi in Hepes-buffered solutions were acid loaded, pHi recovered very slowly. Neurons with a relatively high initial pHi recovered rapidly. The rate constant for the exponential pHi recovery increased with initial pHi. All pHi recoveries required Na+. 4. Both for neurons with a relatively high (> or = 7.05) and a relatively low (< 7.05) initial pHi, net acid extrusion rates (Jtotal = dpHi/dt x buffering power) decreased linearly with increasing pHi. Compared with the line for neurons with a relatively low initial pHi, that for neurons with a relatively high pHi had a significantly greater slope and was alkaline shifted by 0.6-0.7 pH units. 5. Removing external Na+ in the absence of CO2/HCO3- caused pHi to decrease by approximately 0.3 in neurons with a relatively low initial pHi, and by approximately 0.5 in neurons with a relatively high initial pHi. This initial acidification was followed by a slower, partial pHi recovery in approximately 32% of neurons with a relatively low initial pHi, but only approximately 14% of neurons with a relatively high pHi. 6. When exposed to CO2/HCO3-, all neurons initially acidified. Neurons with a relatively low initial pHi recovered to a pHi approximately 0.2 pH units greater than the initial value. Among neurons with higher initial pHi values, some did not recover at all, whereas others recovered to a value similar to or above the initial pHi. On average, the final CO2/HCO3- pHi for neurons with a relatively high initial pHi was similar to the pHi in Hepes buffer. Neurons with a relatively high pHi in Hepes buffer continued to be more alkaline (by approximately 0.2 pH units) in CO2/HCO3-. 7. When neurons with a relatively high initial pHi in Hepes (> or = 7.25) were exposed to CO2/HCO3- and then acid loaded, Jtotal values were more than twice the highest values observed in neurons with lower initial pHi values. Neurons with a moderate initial pHi in Hepes (7.05-7.24) had Jtotal values, at comparable pHi values, that were approximately 2-fold greater than for neurons with a relatively low initial pHi (< 7.05). 8. Thus, freshly isolated CA1 neurons of both mature and immature rats have a wide range of acid-base properties. Those with higher initial pHi values in a Hepes buffer tend to have greater Jtotal values in both Hepes and CO2/HCO3-, and tend to have higher steady-state pHi values in CO2/HCO3-.

Use of BCECF and propidium iodide to assess membrane integrity of acutely isolated CA1 neurons from rat hippocampus.

We used 2 fluorescent dyes, 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and propidium iodide (PI), to assess the membrane integrity of neurons acutely isolated from the CA1 region of the rat hippocampus. Exciting BCECF at a relatively pH-insensitive wavelength (440 nm), or exciting PI at 490 nm, we quantitatively recorded, in real time and in single cells, the rate constants for BCECF loss (-k440) and PI uptake (k490). We found that approximately 98% of intracellular BCECF is rapidly released by applying 0.01% saponin. In neurons not treated with saponin, rate constants for BCECF loss and PI uptake typically were 1% min-1 or less under control conditions, in the presence of NH3/NH4+ and in the absence of Na+. However, in a small number of neurons, the rate constant for BCECF loss increased markedly (-k440 > 5% min-1), while pHi approached pHo, suggesting that the plasma membrane spontaneously became leaky. When neurons were progressively swollen in hypotonic solutions, rates constants for BCECF loss and PI uptake generally were affected minimally unless osmolality was decreased to approximately 75 mOsmol/kg. Treating neurons with 0.001% saponin caused an increase in PI uptake rate only in a minority of neurons, whereas in most experiments a similar treatment caused -k440 for BCECF to exceed 5% min-1, and led to a rapid deterioration of the pH gradient across the cell membrane. At even lower saponin levels (0.0005-0.0007%), we observed a much slower deterioration of pHi, which occurred at low rates of BCECF loss (-k440 = approximately 3% min-1). We conclude that computing rate constants for BCECF loss and PI uptake may be useful for assessing neuronal health, and that BCECF loss may be more sensitive to cell damage than PI uptake.

Out-of-equilibrium CO2/HCO3- solutions and their use in characterizing a new K/HCO3 cotransporter.

In typical physiological solutions, CO2 is in equilibrium with HCO3- and H+ (CO2 + H2O<==>HCO3- +H+). Because one cannot independently alter CO2 and HCO3- concentrations and pH, it is impossible to distinguish between the effects of CO2 and HCO3- on physiological processes. Here we describe a continuous-flow, rapid-mixing approach for generating out-of-equilibrium CO2/HCO3- solutions with a physiological pH and CO2 (but little HCO3-), or pH and HCO3- (but little CO2). We have exploited these out-of-equilibrium solutions to introduce HCO3- exclusively to either the outside or inside of a squid giant axon, and verify the presence of a new K/HCO3 cotransporter. The out-of-equilibrium approach could be useful in a variety of applications for independently controlling CO2 and HCO3- concentrations and pH.