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

Bevensee MO, Apkon M, Boron WF - J. Gen. Physiol. (1997)

Bottom Line: Furthermore, the pH decrease elicited by external Na removal does not require external Cl.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, and Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
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.

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Hippocampal astrocytes could have any of three known  Na+-driven HCO3− transport mechanisms.
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Figure 1: Hippocampal astrocytes could have any of three known Na+-driven HCO3− transport mechanisms.

Mentions: As described in the accompanying paper (Bevensee et al., 1997), exposing rat hippocampal astrocytes to CO2/ HCO3− causes pHi to decrease initially, due to the influx of CO2, and then generally to increase to a value higher than the initial one prevailing in the nominal absence of CO2/HCO3−. Because this pHi increase is blocked by the HCO3−-transport inhibitors 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS)1 and 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), and requires external Na+, we concluded that the astrocytes have a Na+-driven HCO3− transporter. The data are consistent with the presence of one or more of three HCO3− transporters known to exist in other cells (Fig. 1): (a) a Na+-driven Cl-HCO3 exchanger, (b) an electroneutral Na/HCO3 cotransporter with a Na+:HCO3− stoichiometry of 1:1, and (c) an electrogenic Na/HCO3 cotransporter with a Na+:HCO3− stoichiometry of 1:2. Theoretically, a fourth possibility is the electrogenic NaHCO3 cotransporter with a 1:3 stoichiometry, as exists in renal proximal tubules (Boron and Boulpaep, 1983; Soleimani et al., 1987). However, given the ion and voltage gradients likely to prevail in an astrocyte, this 1:3 cotransporter would almost certainly mediate net HCO3− efflux, and not the influx necessary to account for the observed CO2/HCO3−-induced alkalinization.


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

Bevensee MO, Apkon M, Boron WF - J. Gen. Physiol. (1997)

Hippocampal astrocytes could have any of three known  Na+-driven HCO3− transport mechanisms.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2229371&req=5

Figure 1: Hippocampal astrocytes could have any of three known Na+-driven HCO3− transport mechanisms.
Mentions: As described in the accompanying paper (Bevensee et al., 1997), exposing rat hippocampal astrocytes to CO2/ HCO3− causes pHi to decrease initially, due to the influx of CO2, and then generally to increase to a value higher than the initial one prevailing in the nominal absence of CO2/HCO3−. Because this pHi increase is blocked by the HCO3−-transport inhibitors 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS)1 and 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid (SITS), and requires external Na+, we concluded that the astrocytes have a Na+-driven HCO3− transporter. The data are consistent with the presence of one or more of three HCO3− transporters known to exist in other cells (Fig. 1): (a) a Na+-driven Cl-HCO3 exchanger, (b) an electroneutral Na/HCO3 cotransporter with a Na+:HCO3− stoichiometry of 1:1, and (c) an electrogenic Na/HCO3 cotransporter with a Na+:HCO3− stoichiometry of 1:2. Theoretically, a fourth possibility is the electrogenic NaHCO3 cotransporter with a 1:3 stoichiometry, as exists in renal proximal tubules (Boron and Boulpaep, 1983; Soleimani et al., 1987). However, given the ion and voltage gradients likely to prevail in an astrocyte, this 1:3 cotransporter would almost certainly mediate net HCO3− efflux, and not the influx necessary to account for the observed CO2/HCO3−-induced alkalinization.

Bottom Line: Furthermore, the pH decrease elicited by external Na removal does not require external Cl.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, and Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus