Intracellular pH Regulation in Cultured Astrocytes from Rat Hippocampus I. Role of HCO

We studied the regulation of intracellular pH (pH i ) in single cultured astrocytes passaged once from the hippocampus of the rat, using the dye 2 9 ,7 9 -biscarboxyethyl-5,6-carboxyfluorescein (BCECF) to monitor pH i . Intrinsic buffering power ( b I ) was 10.5 mM (pH unit) 2 1 at pH i 7.0, and decreased linearly with pH i ; the best-fit line to the data had a slope of 2 10.0 mM (pH unit) 2 2 . In the absence of HCO 3 2 , pH i 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 i 6.05. The ethylisopropylamiloride-sensitive component of acid extrusion fell linearly with pH i . Acid extrusion was inhibited 68% (pH i 6.23) by substituting Li 1 for Na 1 in the bath solution. Switching from a CO 2 /HCO 3 2 -free to a CO 2 /HCO 3 2 -containing bath solution caused mean steady state pH i to increase from 6.82 to 6.90, due to a Na 1 -driven HCO 3 2 transporter. The HCO 3 2 -induced pH i increase was unaffected by amiloride, but was inhibited 75% (pH i 6.85) by 400 m M 4,4 9 -diisothiocyanatostilbene-2,2 9 -disulfonic acid (DIDS), and 65% (pH i 6.55–6.75) by pretreating astrocytes for up to z 6.3 h with 400 m M 4-acetamide-4 9 -isothiocyanatostilbene-2,2 9 -disulfonic acid (SITS). The CO 2 /HCO 3 2 -induced pH i increase was blocked when external Na 1 was replaced with N -methyld -glucammonium (NMDG 1 ). In the presence of HCO 3 2 , the Na 1 -driven HCO 3 2 transporter contributed to the pH i recovery from an acid load. For example, HCO 3 2 shifted the plot of acid-extrusion rate vs. pH i by 0.15–0.3 pH units in the alkaline direction. Also, with Na-H exchange inhibited by amiloride, HCO 3 2 increased acid extrusion 3.8-fold (pH i 6.20). When astrocytes were acid loaded in amiloride, with Li 1 as the major cation, HCO 3 2 failed to elicit a substantial increase in pH i . Thus, Li 1 does not appear to substitute well for Na 1 on the HCO 3 2 transporter. We conclude that an amiloride-sensitive Na-H exchanger and a Na 1 -driven HCO 3 2 transporter are the predominant acid extruders in astrocytes. key words: H 1 concentration • acid–base transport • glia • nervous system • Na-H exchanger i n t r o d u c t i o n It is well established that the pH of the brain extracellular fluid (pH ECF ) 1 can influence neuronal activity (for reviews, see Chesler and Kaila, 1992; Ransom, 1992), especially because many ion channels are sensitive to changes in extracellular pH (pH o ) (for reviews see Moody, 1984; Chesler, 1990). For example, the open probability of the N -methyld -aspartate-activated channel decreases at progressively lower pH o values, with an apparent pK in the range 6.7–7.3 (Tang et al., 1990; Traynelis and Cull-Candy, 1990). The relationship between changes in pH ECF and neuronal activity is complicated, however, because electrical activity itself can alter the pH of the brain extracellular fluid. The changes in pH ECF elicited by neuronal firing are caused by the transport of acid–base equivalents across the plasma membrane of neurons and/or glia cells. This acid–base transport will have two effects. First, it obviously will affect the pH i of the cells doing the transport, as well as the pH ECF in the microenvironment. Second, because acid–base transporters on neighboring cells generally are sensitive to such pH ECF changes, there will be indirect effects on the pH i of these neighboring cells. pH ECF is thus in the position to mediate a complex interaction among neurons and glial cells. Indeed, the glial cells are thought to play a key role in regulating ECF composition, including pH ECF , and the acid–base transporters of both invertebrate and mammalian glial cells are capable of modulating pH ECF (see review by Deitmer and Rose, 1996). One such transporter thought to play an important role in regulating pH ECF is the electrogenic Na/HCO 3 cotransporter. A similar transporter was first described in the cells of the proximal tuPortions of this work have been published in preliminary form (Bevensee, M.O., R.A. Weed, and W.F. Boron. 1993. FASEB J. 7: A 186.). Address correspondence to Walter F. Boron, Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Fax: 203-785-4951; E-mail: [email protected] 1 Abbreviations used in this paper: BCECF-AM, acetoxymethyl ester of the pH-sensitive dye 2 9 ,7 9 -biscarboxyethyl-5,6-carboxyfluorescein; DIDS, 4,4 9 -diisothiocyanatostilbene-2,2 9 -disulfonic acid; EIPA, ethylisopropylamiloride; NMDG 1 , N -methyld -glucammonium; pH ECF , pH of the brain extracellular fluid; SITS, 4-acetamido-4 9 -isothiocyanatostilbene2,2 9 -disulfonic acid. on A ril 2, 2008 w w w .jg.org D ow nladed fom on S etem er 3, 2016 D ow nladed fom Published October 1, 1997