Biomedical subjects

W F Boron

Publications and source records attributed to W F Boron.

At least 37 records · Page 2Linked to original sources

PubMed1999

Expression and distribution of the Na(+)-HCO(-)(3) cotransporter in human pancreas.

The cellular mechanisms of HCO(-)(3) secretion in the human pancreas are unclear. Expression of a Na(+)-HCO(-)(3) cotransporter (NBC) mRNA has been observed recently, but the distribution and physiological role of the NBC protein are not known. Here we examined the expression and localization of NBC in human pancreas by Northern blot, immunoblot, and immunofluorescence microscopy. Rat kidney NBC probes detected a single 9.5-kb band by Northern blot. On immunoblots, two polyclonal antisera directed against different epitopes of rat kidney NBC identified a single approximately 130-kDa protein. In cryosections of normal human pancreas, both antisera labeled basolateral membranes of large, morphologically identifiable ducts and produced a distinct labeling pattern in the remainder of the parenchyma. In double-labeling experiments, NBC immunoreactivity in the parenchyma colocalized with the Na(+)-K(+) pump, a basolateral marker. In contrast, NBC and cystic fibrosis transmembrane conductance regulator, an apical membrane marker, were detected within the same histological structures but at different subcellular localizations. The NBC antisera did not label acinar or islet cells. Our observations suggest that secretion of HCO(-)(3) by human pancreatic duct cells involves the basolateral uptake of Na(+) and HCO(-)(3) via NBC, an electrogenic Na(+)-HCO(-)(3) cotransporter.

Aged↗

PubMed1999

Immunolocalization of anion exchanger AE2 and Na(+)-HCO(-)(3) cotransporter in rat parotid and submandibular glands.

Salivary glands secrete K(+) and HCO(-)(3) and reabsorb Na(+) and Cl(-), but the identity of transporters involved in HCO(-)(3) transport remains unclear. We investigated localization of Cl(-)/HCO(-)(3) exchanger isoform AE2 and of Na(+)-HCO(-)(3) cotransporter (NBC) in rat parotid gland (PAR) and submandibular gland (SMG) by immunoblot and immunocytochemical techniques. Immunoblotting of PAR and SMG plasma membranes with specific antibodies against mouse kidney AE2 and rat kidney NBC revealed protein bands at approximately 160 and 180 kDa for AE2 and approximately 130 kDa for NBC, as expected for the AE2 full-length protein and consistent with the apparent molecular mass of NBC in several tissues other than kidney. Immunostaining of fixed PAR and SMG tissue sections revealed specific basolateral staining of PAR acinar cells for AE2 and NBC, but in SMG acinar cells only basolateral AE2 labeling was observed. No AE2 expression was detected in any ducts. Striated, intralobular, and main duct cells of both glands showed NBC expression predominantly at basolateral membranes, with some cells being apically stained. In SMG duct cells, NBC staining exhibited a gradient of distribution from basolateral localization in more proximal parts of the ductal tree to apical localization toward distal parts of the ductal tree. Both immunoblotting signals and immunostaining were abolished in preabsorption experiments with the respective antigens. Thus the mechanisms of fluid and anion secretion in salivary acinar cells may be different between PAR and SMG, and, because NBC was detected in acinar and duct cells, it may play a more important role in transport of HCO(-)(3) by rat salivary duct cells than previously believed.

Animals↗

PubMed1999

Immunolocalization of the electrogenic Na+-HCO-3 cotransporter in mammalian and amphibian kidney.

Electrogenic cotransport of Na+ and HCO-3 is a crucial element of HCO-3 reabsorption in the renal proximal tubule (PT). An electrogenic Na+-HCO-3 cotransporter (NBC) has recently been cloned from salamander and rat kidney. In the present study, we generated polyclonal antibodies (pAbs) to NBC and used them to characterize NBC on the protein level by immunochemical methods. We generated pAbs in guinea pigs and rabbits by immunizing with a fusion protein containing the carboxy-terminal 108 amino acids (amino acids 928-1035) of rat kidney NBC (rkNBC). By indirect immunofluorescence microscopy, the pAbs strongly labeled HEK-293 cells transiently expressing NBC, but not in untransfected cells. By immunoblotting, the pAbs recognized a approximately 130-kDa band in Xenopus laevis oocytes expressing rkNBC, but not in control oocytes injected with water or cRNA for the Cl-/HCO-3 exchanger AE2. In immunoblotting experiments on renal microsomes, the pAbs specifically labeled a major band at approximately 130 kDa in both rat and rabbit, as well as a single approximately 160-kDa band in salamander kidney. By indirect immunofluorescence microscopy on 0.5-micrometer cryosections of rat and rabbit kidneys fixed in paraformaldehyde-lysine-periodate (PLP), the pAbs produced a strong and exclusively basolateral staining of the PT. In the salamander kidney, the pAbs labeled only weakly the basolateral membrane of the PT. In contrast, we observed strong basolateral labeling in the late distal tubule, but not in the early distal tubule. The specificity of the pAbs for both immunoblotting and immunohistochemistry was confirmed in antibody preabsorption experiments using either the fusion protein used for immunization or similarly prepared control fusion proteins. In summary, we have developed antibodies specific for NBC, determined the apparent molecular weights of rat, rabbit, and salamander kidney NBC proteins, and described the localization of NBC within the kidney of these mammalian and amphibian species.

Ambystoma↗

PubMed1998

Effect of expressing the water channel aquaporin-1 on the CO2 permeability of Xenopus oocytes.

It is generally accepted that gases such as CO2 cross cell membranes by dissolving in the membrane lipid. No role for channels or pores in gas transport has ever been demonstrated. Here we ask whether expression of the water channel aquaporin-1 (AQP1) enhances the CO2 permeability of Xenopus oocytes. We expressed AQP1 in Xenopus oocytes by injecting AQP1 cRNA, and we assessed CO2 permeability by using microelectrodes to monitor the changes in intracellular pH (pHi) produced by adding 1.5% CO2/10 mM HCO3- to (or removing it from) the extracellular solution. Oocytes normally have an undetectably low level of carbonic anhydrase (CA), which eliminates the CO2 hydration reaction as a rate-limiting step. We found that expressing AQP1 (vs. injecting water) had no measurable effect on the rate of CO2-induced pHi changes in such low-CA oocytes: adding CO2 caused pHi to fall at a mean initial rate of 11.3 x 10(-4) pH units/s in control oocytes and 13.3 x 10(-4) pH units/s in oocytes expressing AQP1. When we injected oocytes with water, and a few days later with CA, the CO2-induced pHi changes in these water/CA oocytes were more than fourfold faster than in water-injected oocytes (acidification rate, 53 x 10(-4) pH units/s). Ethoxzolamide (ETX; 10 microM), a membrane-permeant CA inhibitor, greatly slowed the pHi changes (16.5 x 10(-4) pH units/s). When we injected oocytes with AQP1 cRNA and then CA, the CO2-induced pHi changes in these AQP1/CA oocytes were approximately 40% faster than in the water/CA oocytes (75 x 10(-4) pH units/s), and ETX reduced the rates substantially (14.7 x 10(-4) pH units/s). Thus, in the presence of CA, AQP1 expression significantly increases the CO2 permeability of oocyte membranes. Possible explanations include 1) AQP1 expression alters the lipid composition of the cell membrane, 2) AQP1 expression causes overexpression of a native gas channel, and/or 3) AQP1 acts as a channel through which CO2 can permeate. Even if AQP1 should mediate a CO2 flux, it would remain to be determined whether this CO2 movement is quantitatively important.

Animals↗

PubMed1998

Effect of PCMBS on CO2 permeability of Xenopus oocytes expressing aquaporin 1 or its C189S mutant.

A recent study on Xenopus oocytes [N. L. Nakhoul, M. F. Romero, B. A. Davis, and W. F. Boron. Am. J. Physiol. 274 (Cell Physiol. 43): C543-548, 1998] injected with carbonic anhydrase showed that expressing aquaporin 1 (AQP1) increases by approximately 40% the rate at which exposing the cell to CO2 causes intracellular pH to fall. This observation is consistent with several interpretations. Overexpressing AQP1 might increase apparent CO2 permeability by 1) allowing CO2 to pass through AQP1, 2) stimulating injected carbonic anhydrase, 3) enhancing the CO2 solubility of the membrane's lipid, or 4) increasing the expression of a native "gas channel." The purpose of the present study was to distinguish among these possibilities. We found that expressing the H2O channel AQP1 in Xenopus oocytes increases the CO2 permeability of oocytes in an expression-dependent fashion, whereas expressing the K+ channel ROMK1 has no effect. The mercury derivative p-chloromercuriphenylsulfonic acid (PCMBS), which inhibits the H2O movement through AQP1, also blocks the AQP1-dependent increase in CO2 permeability. The mercury-insensitive C189S mutant of AQP1 increases the CO2 permeability of the oocyte to the same extent as does the wild-type channel. However, the C189S-dependent increase in CO2 permeability is unaffected by treatment with PCMBS. These data rule out options 2-4 listed above. Thus our results suggest that CO2 passes through the pore of AQP1 and are the first data to demonstrate that a gas can enter a cell by a means other than diffusing through the membrane lipid.

4-Chloromercuribenzenesulfonate↗

PubMed1998

Cloning and functional expression of rNBC, an electrogenic Na(+)-HCO3- cotransporter from rat kidney.

We have recently cloned the renal electrogenic Na(+)-bicarbonate contransporter of the salamander Ambystoma tigrinum (aNBC) (M. F. Romero, M. A. Hediger, E. L. Boulpaep, and W. F. Boron. FASEB J. 10: 89, 1996; and Nature 387: 409-413, 1997). Here we report the cloning of a mammalian homolog of aNBC, named rNBC for rat Na(+)-bicarbonate cotransporter. NBC constitutes the major route for HCO3- reabsorption and assists in Na+ reabsorption across the basolateral membrane of the renal proximal tubule (PT). We used aNBC as a probe to screen a rat kidney cortex cDNA library in lambda gt10 and identified several clones. Each has an initiator Met and a large open-reading frame followed by a 3'-untranslated region of approximately 500 bp. The 7.5-kb mRNA for rNBC is present in kidney, liver, lung, brain, and heart. In situ hybridization with the rNBC probe in the rat kidney revealed staining in the S2 segment of PT. rNBC encodes a protein of 1,035 amino acids, with a predicted molecular mass of 116 kDa. Its deduced amino acid sequence is 86% identical to that of aNBC. Comparison of both the aNBC and rNBC sequences to the GenBank database reveals a low level of amino acid identity (approximately 30%) to the AE family of Cl-/HCO3- exchangers. Injection of rNBC cRNA into Xenopus oocytes leads to expression of an electrogenic Na(+)-HCO3- contransporter that is qualitatively similar to that of aNBC but at a much lower level. Placement of the rNBC cDNA into the context of a Xenopus expression vector produces a substantial increase in rNBC expression. Addition of 1.5% CO2/10 mM HCO3- elicits a hyperpolarization of > 50 mV and a rapid decrease of intracellular pH (pHi), followed by an increase in pHi. Subsequent removal of Na+ in the presence of CO2/HCO3- causes a depolarization of > 50 mV and a concomitant decrease of pHi. Thus rNBC is in the same newly identified family of Na(+)-linked HCO3- transporters as is aNBC.

Amino Acid Sequence↗

PubMed1997

Cloning and characterization of a mammalian proton-coupled metal-ion transporter.

Metal ions are essential cofactors for a wealth of biological processes, including oxidative phosphorylation, gene regulation and free-radical homeostasis. Failure to maintain appropriate levels of metal ions in humans is a feature of hereditary haemochromatosis, disorders of metal-ion deficiency, and certain neurodegenerative diseases. Despite their pivotal physiological roles, however, there is no molecular information on how metal ions are actively absorbed by mammalian cells. We have now identified a new metal-ion transporter in the rat, DCT1, which has an unusually broad substrate range that includes Fe2+, Zn2+, Mn2+, Co2+, Cd2+, Cu2+, Ni2+ and Pb2+. DCT1 mediates active transport that is proton-coupled and depends on the cell membrane potential. It is a 561-amino-acid protein with 12 putative membrane-spanning domains and is ubiquitously expressed, most notably in the proximal duodenum. DCT1 is upregulated by dietary iron deficiency, and may represent a key mediator of intestinal iron absorption. DCT1 is a member of the 'natural-resistance-associated macrophage protein' (Nramp) family and thus its properties provide insight into how these proteins confer resistance to pathogens.

Amino Acid Sequence↗

PubMed1997

The electrogenic Na/HCO3 cotransporter.

The electrogenic Na/HCO3 cotransporter (symporter) is the major HCO3- transporter of the renal proximal tubule (PiT), located at the basolateral membrane (BLM), and also plays a noteworthy role in Na+ reabsorption. HCO3 transporters are important for regulation of intracellular pH (pHi) in most cells and also thereby regulate blood pH. This electrogenic Na/HCO3 cotransporter was first discovered using perfused Ambystoma tigrinum (salamander) renal, proximal tubules. This novel cotransporter mediates the movement of one Na+ ion with several HCO3- ions, making it electrogenic, is blocked by stilbene compounds, but does not depend on intra- or extracellular Cl-. This and similar cotransporters have been found in a number of tissues and cell types. Recently, we used Xenopus-laevis oocytes to expression clone the salamander renal electrogenic Na Bicarbonate Cotransporter (NBC). Using microelectrodes to monitor membrane potential (Vm) and intracellular pH (pHi), we followed oocyte expression after injecting poly (A)+, fractioned poly (A)+, or cRNA. All experimental solutions contained 100 microM ouabain to block the Na+/K+ pump. Our expression assay was to apply 1.5% CO2/10 mM HCO3- (pH 7.5), allow pHi to stabilize from the CO2-induced acidification, and then remove bath Na+. Removing bath Na+ from native oocytes and water-injected controls, hyperpolarized the oocytes by approximately 5 mV and had no effect on pHi. However, for oocytes injected with poly (A)+ RNA, removing Na+ transiently depolarized the cell by approximately 10 mV and caused pHi to decrease; both effects were blocked by 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS) and required HCO3-. Electrophoretic fractionation of the poly (A)+ RNA, enriched the expression signal. From the optimal expression-fraction, we constructed a size-selected cDNA library in pSPORT1. Screening our Ambystoma library yielded a single clone (aNBC). We could detect expression 3 days after injection of NBC cRNA. In aNBC-expressing oocytes, adding CO2/HCO3-elicited a large (> 50mV) and rapid hyperpolarization, followed by a partial relaxation as pHi stabilized. Na+ removal in CO2/HCO3-depolarized the cell by > 40mV and decreased pHi, aNBC encodes a protein of 1035 amino acids with several putative membrane-spanning domains, and has a low level of amino-acid homology (approximately 30% to the AE family of Cl-HCO3 exchangers. aNBC is the first member of a new family of Na(+)-linked HCO3- transporters and, together with the AE family, defines a new superfamily of HCO3- transporters. Using aNBC to screen a rat-kidney cDNA library, we identified a full-length cDNA clone (rNBC), rNBC encodes a protein of 1035 amino acids, is 86% identical to aNBC, and can be functionally expressed in oocytes.

Acid-Base Equilibrium↗

PubMed1997

Expression cloning and characterization of a renal electrogenic Na+/HCO3- cotransporter.

Bicarbonate transporters are the principal regulators of pH in animal cells, and play a vital role in acid-base movement in the stomach, pancreas, intestine, kidney, reproductive system and central nervous system. The functional family of HCO3- transporters includes Cl- -HCO3- exchangers, three Na+/HCO3- cotransporters, a K+/HCO3- cotransporter, and a Na+-driven Cl- -HCO3- exchanger. Molecular information is sparse on HCO3- transporters, apart from Cl- -HCO3- exchangers ('anion exchangers'), whose complementary DNAs were cloned several years ago. Attempts to clone other HCO3- transporters, based on binding of inhibitors, protein purification or homology with anion exchangers, have so far been unsuccessful. Here we monitor the intracellular pH and membrane voltage in Xenopus oocytes to follow the expression of the most electrogenic transporter known: the renal 1:3 electrogenic Na+/HCO3- cotransporter from the salamander Ambystoma tigrinum. We now report the successful cloning and characterization of a cDNA encoding a cation-coupled HCO3- transporter. The encoded protein is 1,035 amino acids long with several potential membrane-spanning domains. We show that when it is expressed in Xenopus oocytes, this protein is electrogenic, Na+ and HCO3- dependent, and blocked by the anion-transport inhibitor DIDS, and conclude that it is the renal electrogenic sodium bicarbonate cotransporter (NBC).

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1997

Symmetry of H+ binding to the intra- and extracellular side of the H+-coupled oligopeptide cotransporter PepT1.

Ion-coupled solute transporters exhibit pre-steady-tate currents that resemble those of voltage-dependent ion channels. These currents were assumed to be mostly due to binding and dissociation of the coupling ion near the extracellular transporter surface. Little attention was given to analogous events that may occur at the intracellular surface. To address this issue, we performed voltage clamp studies of Xenopus oocytes expressing the intestinal H+-coupled peptide cotransporter PepT1 and recorded the dependence of transient charge movements in the absence of peptide substrate on changing intra- (pHi) and extracellular pH (pHo). Rapid steps in membrane potential induced transient charge movements that showed a marked dependence on pHi and pHo. At a pHo of 7.0 and a holding potential (Vh) of -50 mV, the charge movements were mostly inwardly directed, whereas reduction of pHo to below 7.0 resulted in outwardly directed charge movements. When pHi was reduced, inwardly directed charge movements were observed. The data on the voltage dependence of the transient charge movements were fitted by the Boltzmann equation, yielding an apparent valence of 0.65 +/- 0.03 (n = 7). The midpoint voltage (V0.5) of the charge distribution shifted linearly as a function of pHi and pHo. Our results indicate that, as a first approximation, the magnitude and polarity of the transient charge movements depend upon the prevailing H+ electrochemical gradient. We propose that PepT1 has a single proton binding site that is symmetrically accessible from both sides of the membrane and that decreasing the H+ chemical potential (DeltamuH) or increasing the membrane potential (Vm) shifts this binding site from an outwardly to an inwardly facing occluded state. This concept constitutes an important extension of previous kinetic models of ion-coupled solute transporters by including a more detailed description of intracellular events.

Animals↗

PubMed1997

Stoichiometry and pH dependence of the rabbit proton-dependent oligopeptide transporter PepT1.

1. The intestinal H(+)-coupled peptide transporter PepT1, displays a broad substrate specificity and accepts most charged and neutral di- and tripeptides. To study the proton-to-peptide stoichiometry and the dependence of the kinetic parameters on extracellular pH (pHo), rabbit PepT1 was expressed in Xenopus laevis oocytes and used for uptake studies of radiolabelled neutral and charged dipeptides, voltage-clamp analysis and intracellular pH measurements. 2. PepT1 did not display the substrate-gated anion conductances that have been found to be characteristic of members of the Na(+)- and H(+)-coupled high-affinity glutamate transporter family. In conjunction with previous data on the ion dependence of PepT1, it can therefore be concluded that peptide-evoked charge fluxes of PepT1 are entirely due to H+ movement. 3. Neutral, acidic and basic dipeptides induced intracellular acidification. The rate of acidification, the initial rates of the uptake of radiolabelled peptides and the associated charge fluxes gave proton-substrate coupling ratios of 1:1, 2:1 and 1:1 for neutral, acidic and basic dipeptides, respectively. 4. Maximal transport of the neutral and charged dipeptides Gly-Leu, Gly-Glu, Gly-Lys and Ala-Lys occurred at pHo 5.5, 5.2, 6.2 and 5.8, respectively. The Imax values were relatively pHo independent but the apparent affinity (Km(app) values for these peptides were shown to be highly pHo dependent. 5. Our data show that at physiological pH (pHo 5.5-6.0) PepT1 prefers neutral and acidic peptides. The shift in transport maximum for the acidic peptide Gly-Glu to a lower pH value suggests that acidic dipeptides are transported in the protonated form. The shift in the transport maxima of the basic dipeptides to higher pH values may involve titration of a side-chain on the transporter molecule (e.g. protonation of a histidine group). These considerations have led us to propose a model for coupled transport of neutral, acidic and basic dipeptides.

Animals↗

PubMed1997

Sodium kinetics of Na,K-ATPase alpha isoforms in intact transfected HeLa cells.

By participating in the regulation of ion and voltage gradients, the Na-K pump (i.e., Na,K-ATPase) influences many aspects of cellular physiology. Of the four alpha isoforms of the pump, alpha1 is ubiquitous, alpha2 is predominant in skeletal muscle, and alpha3 is found in neurons and the cardiac conduction system. To determine whether the isoforms have different intracellular Na+ affinities, we used the Na+-sensitive dye sodium-binding benzofuran isophthalate (SBFI) to measure pump-mediated Na+ efflux as a function of [Na+]i in human HeLa cells stably transfected with rat Na-K pump isoforms. We Na+-loaded the cells, and then monitored the time course of the decrease in [Na+]i after removing external Na+. All transfected rat alpha subunits were highly ouabain resistant: the alpha1 isoform is naturally resistant, whereas the alpha2 and alpha3 isoforms had been mutagenized to render them resistant. Thus, the Na+ efflux mediated by endogenous and transfected pumps could be separated by studying the cells at low (1 microM) and high (4 mM) ouabain concentrations. We found that the apparent Km for Na+ efflux attributable to the native human alpha1 isoform was 12 mM, which was similar to the Km of rat alpha1. The alpha2 and alpha3 isoforms had apparent Km's of 22 and 33 mM, respectively. The cells expressing alpha3 had a high resting [Na+]i. The maximal activity of native alpha1 in the alpha3-transfected cells was only approximately 56% of native alpha1 activity in untransfected HeLa cells, suggesting that transfection with alpha3 led to a compensatory decrease in endogenous alpha1 pumps. We conclude that the apparent Km(Na+) for rat Na-K pump isoforms increases in the sequence alpha1 < alpha2 < alpha3. The alpha3 isoform may be suited for handling large Na+ loads in electrically active cells.

Animals↗

PubMed1997

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.

Animals↗

PubMed1997

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.

Animals↗

PubMed1997

Intracellular Cl- dependence of Na-H exchange in barnacle muscle fibers under normotonic and hypertonic conditions.

We previously showed that shrinking a barnacle muscle fiber (BMF) in a hypertonic solution (1,600 mosM/kg) stimulates an amiloride-sensitive Na-H exchanger. This activation is mediated by a G protein and requires intracellular Cl-. The purpose of the present study was to determine (a) whether Cl- plays a role in the activation of Na-H exchange under normotonic conditions (975 mosM/kg), (b) the dose dependence of [Cl-]i for activation of the exchanger under both normo- and hypertonic conditions, and (c) the relative order of the Cl-- and G-protein-dependent steps. We acid loaded BMFs by internally dialyzing them with a pH-6.5 dialysis fluid containing no Na+ and 0-194 mM Cl-. The artificial seawater bathing the BMF initially contained no Na+. After dialysis was halted, adding 50 mM Na+ to the artificial seawater caused an amiloride-sensitive pHi increase under both normo- and hypertonic conditions. The computed Na-H exchange flux (JNa-H) increased with increasing [Cl-]i under both normo- and hypertonic conditions, with similar apparent Km values ( approximately 120 mM). However, the maximal JNa-H increased by nearly 90% under hypertonic conditions. Thus, activation of Na-H exchange at low pHi requires Cl- under both normo- and hypertonic conditions, but at any given [Cl-]i, JNa-H is greater under hyper- than normotonic conditions. We conclude that an increase in [Cl-]i is not the primary shrinkage signal, but may act as an auxiliary shrinkage signal. To determine whether the Cl--dependent step is after the G-protein-dependent step, we predialyzed BMFs to a Cl--free state, and then attempted to stimulate Na-H exchange by activating a G protein. We found that, even in the absence of Cl-, dialyzing with GTPgammaS or AlF3, or injecting cholera toxin, stimulates Na-H exchange. Because Na-H exchange activity was absent in control Cl--depleted fibers, the Cl--dependent step is at or before the G protein in the shrinkage signal-transduction pathway. The stimulation by AlF3 indicates that the G protein is a heterotrimeric G protein.

Aluminum Compounds↗

PubMed1997

Motor responses of cultured rat cerebral vascular smooth muscle cells to intra- and extracellular pH changes.

Alkalizing perivascular fluid constricts, whereas acidification dilates, cerebral arterioles. It is not known whether vascular smooth muscle cells (VSMCs), endothelium, or neuronal elements sense pH changes. We hypothesized that VSMCs themselves transduce extracellular pH (pHo) changes. We examined the motor responses of cultured adult rat middle cerebral arterial VSMCs during pHo and intracellular pH (pHi) changes. Motor responses were inferred from the deformation pattern of a silicone substratum, dimethylpolysiloxane, which wrinkles as cells contract. pHi was measured with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Cultured VSMCs retained motor responses to vasoconstrictors (5-hydroxytryptamine and K+), and to sodium nitroprusside, which were typical of intact arterioles. VSMCs contracted with increasing and relaxed with decreasing pHo. Hypocapnia contracted VSMCs when the pHo increased, and hypercapnia relaxed VSMCs when the pHo decreased. However, at a constant pHo, changes in PCO2 caused opposite responses despite equivalent changes in pHi. Thus VSMCs contract with increased pHo and relax with decreased pHo just as intact arterioles do. These responses do not reflect changes in pHi or PCO2. pHi changes paradoxically alter VSMC tone in the direction opposite that caused by pHo changes.

Animals↗

PubMed1996

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-.

Aging↗

PubMed1996

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.

Acids↗