Intracellular acidification induced by passive and active transport of ammonium ions in astrocytes

Tavarekere N. Nagaraja and Neville Brookes

Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21202

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We describe an unconventional response of intracellular pH to NH4Cl in mouse cerebral astrocytes. Rapid alkalinization reversed abruptly to be replaced by an intense sustained acidification in the continued presence of NH4Cl. We hypothesize that high-velocity NH+4 influx persisted after the distribution of ammonia attained steady state. From the initial rate of acidification elicited by 1 mM NH4Cl in bicarbonate-buffered solution, we estimate that NH+4 entered at a velocity of at least 31.5 nmol · min-1 · mg protein-1. This rate increased with NH4Cl concentration, not saturating at up to 20 mM NH4Cl. Acidification was attenuated by raising or lowering extracellular K+ concentration. Ba2+ (50 µM) inhibited the acidification rate by 80.6%, suggesting inwardly rectifying K+ channels as the primary NH+4 entry pathway. Acidification was 10-fold slower in rat hippocampal astrocytes, consistent with the difference reported for K+ flux in vitro. The combination of Ba2+ and bumetanide prevented net acidification by 1 mM NH4Cl, identifying the Na+-K+-2Cl- cotransporter as a second NH+4 entry route. NH+4 entry via K+ transport pathways could impact "buffering" of ammonia by astrocytes and could initiate the elevation of extracellular K+ concentration and astrocyte swelling observed in acute hyperammonemia.

potassium channels; sodium-potassium-chloride cotransport; hyperammonemia; intracellular pH; mouse; rat

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

MUCH OF THE AMMONIA produced in the brain, or reaching the brain by traversing the blood-brain barrier, is trapped in the form of glutamine after entry into astrocytes (14), where glutamine synthase activity is primarily located (32). Uncharged NH3 is relatively lipid soluble and, in general, diffuses freely across cell membranes (14). However, <2% of total ammonia is uncharged at pH 7.4, assuming a pKa of 9.2. The much more abundant charged species NH+4 has a hydrated ionic radius similar to K+ and is known to share K+ transport pathways (17, 20). Thus transmembrane fluxes of NH+4 also are appreciable. The charged and uncharged species have distinctly different effects on pH in compartments between which they migrate. When NH3 is the predominant form entering a cell, the association of NH3 with H+ to form NH+4 tends to alkalinize the cytoplasm, whereas predominant entry of NH+4 can acidify by reversing this reaction. At physiological ammonia concentrations of 0.1-0.2 mM, such transmembrane fluxes may present a load to pH regulatory mechanisms without appreciably altering intracellular pH (pHi) (9). However, significant pH changes are detected at pathophysiological blood ammonia levels of >= 0.5 mM (11, 22, 40). In this report, the term "ammonia" is used to indicate the total of the charged and uncharged species, whereas the separate species are identified by their chemical formulas.

The transport of ammonia in the individual cell types of the brain has received little direct attention. However, use of the ammonia-prepulse technique (3) to create an intracellular acid load is a common maneuver in studies of pH regulation in brain cells. In such studies the responses observed in neurons and glia have generally conformed to the pattern exhibited by many other cell types (7, 37). A high extracellular concentration of NH4Cl (usually 20 mM) elevates pHi as NH3 rapidly enters the cell and is protonated. After dissipation of the inward concentration gradient of NH3, the new plateau level of pHi begins to decline slowly in the continued presence of NH4Cl. A slow influx of NH+4 is believed to contribute to this decline (3). Washout of NH4Cl from the bathing solution then induces a large intracellular acid load as NH3 diffuses outward and intracellular NH+4 dissociates to form protons. This commonly observed pattern is consistent with the view that diffusion of NH3 is much faster than transport of NH+4. As a consequence, it is believed that brain cells are unable to maintain a concentration gradient of NH3. According to this view, the distribution of total ammonia across cell membranes is determined solely by its pKa and the values of pH in the extracellular and intracellular compartments (3, 14, 36, 42).

The present study arose from the observation of an unconventional response of pHi to 20 mM NH4Cl in primary cultures of mouse cerebral astrocytes. The response began with a typical alkalinization, but this reversed abruptly within a few seconds to be replaced by an intense sustained acidification in the continued presence of the NH4Cl. There is a recent preliminary report of ammonia-induced acidification also in glia acutely isolated from the honey bee drone retina (13). An acidifying response to ammonia has been described and explored extensively in the thick ascending limb of Henle's loop, where it is attributable to unusually rapid transport of NH+4 via K+ transport pathways, especially across the apical membrane of the epithelial cells (16, 17, 24). Here we have characterized the effects of NH4Cl on pHi in mouse astrocytes, and we have attempted to interpret these effects in terms of the fluxes of NH3 and NH+4. We used selective inhibitors of K+ transport to identify entry routes for NH+4. Comparative measurements were made in rat astrocyte cultures, which are known to exhibit a membrane permeability for K+ that is much lower than in mouse astrocyte cultures (45, 46). The membrane transport of NH+4 is likely to impact the regulation of pH and the distribution of K+ in astrocytes, particularly in metabolic disorders associated with hyperammonemia.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Astrocyte culture. Cell cultures of astrocytes were prepared from the cerebral hemispheres of 1-day-old outbred mice (CD-1, Charles River) or the hippocampi of 1-day-old outbred rats (CD, Charles River), as described previously in detail (10). The cultures were grown to confluence in 35-mm dishes each containing a 9 × 31-mm glass coverslip. They were used when 3-6 wk old.

Measurement of pHi. The coverslip cultures were transferred to N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-tris(hydroxymethyl)aminomethane (Tris)-buffered salt solution (HTB) containing (in mM) 150 NaCl, 3 KCl, 2 CaCl2, 0.8 MgCl2, 5 D-glucose, and 10 HEPES. The pH of the solution was adjusted to 7.4 or other required value with Tris base, and the osmolarity was raised to 315 mosM with mannitol. K+ concentration ([K+]) was adjusted by equimolar substitution of KCl for NaCl. The astrocytes were loaded with the fluorescent pHi indicator 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) by incubation with a solution of its acetoxymethyl ester (2 µM; Calbiochem, San Diego, CA). The coverslip culture was then washed, mounted in a fluorometric cuvette, and allowed to equilibrate for 10 min in HTB at 35°C. Intracellular BCECF fluorescence was monitored in ratio mode by alternating the excitation wavelength between 500 and 440 nm and measuring emitted fluorescence at 530 nm. The sampling rate of 1 Hz could not fully resolve the peak of initial alkalinization in response to NH4Cl when it was followed by rapid acidification. Consequently, the reported amplitudes of alkalinization may variably underestimate actual peak values, depending on the subsequent rate of acidification. Calibration of the fluorometric signal and estimation of absolute pHi are considered in detail in Calibration of BCECF.

In some experiments, physiological bicarbonate buffering was simulated using a modified Earle's balanced salt solution (EBS) prepared by omitting HEPES-Tris from the HTB formulation (see above) and substituting 26 mM NaHCO3 plus 1 mM NaH2PO4 for 27 mM NaCl. This solution was bubbled with humidified 5% CO2-95% O2 for at least 1 h at 35°C before use, then the humidified gas stream was directed into the opening of the cuvette. When it was required to remove CO2/HCO-3 from the solution, HTB was similarly gassed with 100% O2.

NH4Cl and other reagents were added in 10 µl of 200-fold concentrate (dissolved in water or dimethyl sulfoxide) to 2 ml of stirred solution in the disposable fluorometric cuvette.

Calibration of BCECF. Questions have been raised about the adequacy of the nigericin-K+ method of Thomas et al. (43) for calibrating the fluorometric signal ratio obtained with BCECF. Nett and Deitmer (30) reported that the ion-sensitive microelectrode and BCECF techniques measured similar shifts of pHi (Delta pHi) in leech glia. However, the absolute value of pHi given by nigericin-K+ calibration of BCECF was 0.12 pH unit higher than the microelectrode measurement. Similarly, Boyarsky et al. (6) found that nigericin-K+-calibrated estimates of resting pHi in BCECF-loaded smooth muscle cells were ~0.2 pH unit higher than the value predicted by a bracketing technique using "null solutions." These null solutions are mixtures of membrane-permeant weak acid and weak base designed to elicit zero Delta pHi at a theoretically specified value of pHi.

Our previous studies (9, 10, 29) used a hybrid approach to calibration. For estimation of Delta pHi, the nigericin-K+ method was used (1-10 µM nigericin-125 mM K+), but 1 µM gramicidin was included in some experiments. On the other hand, estimates of the absolute value of pHi were obtained by adding 1 µM nigericin plus 1 µM gramicidin to collapse H+ gradients at the end of each experiment and assigning to the steady-state fluorometric signal ratio the pH value of the cuvette solution measured directly with a pH electrode. As noted by Boyarsky et al. (5), abolishing the [K+] gradient by means other than nigericin (in the present case, by means of gramicidin) should allow nigericin to equalize pHi and extracellular pH (pHo). However, the questions raised about calibration necessitated a reexamination of the effectiveness of the combination of nigericin and gramicidin.

Figure 1B shows the effect of gramicidin on the nigericin-K+ calibration plot, compared in the same culture. Addition of gramicidin produced a downward correction of 0.14 ± 0.005 (SE) pH unit (n = 5) at pH 7.4 but no effect below pH 6.4. This result is consistent with the finding of Boyarsky et al. (5) that the error introduced by nigericin-K+ calibration is absent at pH 6.0 and increases with pH. The recordings in Fig. 1A show the effects of 7.2-null and 7.0-null solutions of trimethylamine propionate (see Table 5 of Ref. 6) on resting pHi. The small alkalinizing response to 7.2-null and the acidifying response to 7.0-null indicate that 7.2 > pHi > 7.0. Estimates of pHi were obtained from the same recordings by assigning the pH of the cuvette solution to the final steady-state ratio reached after adding nigericin plus gramicidin and using the slope of the nigericin-gramicidin-K+ calibration plot to calculate initial resting pHi. The mean of these estimates was 7.10 ± 0.006 (n = 8), in agreement with the null bracketing result and also with the previously published means of 7.11 (10) and 7.07 (29) obtained under the same conditions, i.e., HTB (pH 7.4) equilibrated with air at 35°C. This evidence indicates that the inclusion of gramicidin in calibration procedures for the present and previous studies of astrocytes substantially corrected the error associated with the nigericin-K+ method.


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Fig. 1.   Calibration of fluorometric signal ratio obtained with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). A: effects on intracellular pH (pHi) of pH 7.2-null (left trace) and pH 7.0-null (right trace) solutions of trimethylamine propionate (6), each followed by addition of 5 µM nigericin + 1 µM gramicidin to collapse transmembrane H+ gradient. Upward deflection of trace indicates increasing pHi. Transient acidification elicited by nigericin + gramicidin is assumed to reflect an initial inflow of H+ before dissipation of membrane potential. Extracellular pH was constant at 7.4 throughout. B: plots of signal ratio given by BCECF-loaded astrocytes responding to imposed changes in pH of HEPES-Tris-buffered salt solution (HTB) containing 5 µM nigericin-125 mM K+, with and without 1 µM gramicidin. Validity of calibration depends on pHi and extracellular pH being equalized (see MATERIALS AND METHODS). Linear regressions are shown for data obtained from 1 culture representative of 3 experiments yielding similar results.

Statistics. Values are means ± SE for the number of coverslip cultures (n). The significance of differences was determined by analysis of variance using the Newman-Keuls test for multiple comparisons.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Figure 2 illustrates the unusual effect of NH4Cl on pHi in mouse astrocytes. Shown superimposed is the response to an equal concentration of methylamine hydrochloride, representing a more typical pattern of response to an acute intracellular alkaline load. In the case of methylamine, rapid intracellular alkalinization induced by influx of the membrane-permeant base was followed by a slow relaxation of pHi toward the initial resting value. The rate of this slow decline of pHi may reflect metabolic production of acid, regulatory outward transport of HCO-3 (12), and slow entry of the relatively impermeant charged base (4). By contrast, the alkalinization produced by NH4Cl was transient, because it was superseded by a rapid acidification leading to a sustained reduction of pHi below the initial resting value.


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Fig. 2.   Superimposed typical responses of pHi to methylamine hydrochloride (CH3NH3Cl) or NH4Cl (added at arrow) in mouse cerebral astrocytes. Bathing solution was HTB (pH 7.4) equilibrated with air at 35°C. Methylamine response is reproduced from Fig. 3B of Ref. 29.

Concentration dependence of the effect of NH4Cl on pHi in mouse astrocytes. The responses of pHi to 0.25-20 mM NH4Cl shown in Fig. 3 were measured in nominally HCO-3-free solution (HTB in air) to reduce the buffering power of the cytoplasm and thus amplify effects on pHi. The initial alkalinization in mouse astrocytes peaked within the first 5 s, and its amplitude declined to the detection threshold when the concentration of NH4Cl added was <1 mM. The mean amplitude of the transient alkalinization in response to 1 mM NH4Cl was 0.056 ± 0.002 pH unit (n = 57, cumulative mean). As noted in MATERIALS AND METHODS, this is likely to be an underestimate because of the limitation on data sampling rate.


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Fig. 3.   Concentration dependence of effect of NH4Cl on pHi in mouse and rat astrocytes. Plotted against NH4Cl concentration (logarithmic abscissa) are peak increase in pHi (alkalinization, positive Delta pHi) relative to initial resting value (A), initial rate of acidification (negative dpHi/dt expressed as pH units/min) after peak of alkalinization (B), and peak decrease in pHi (acidification, negative Delta pHi) relative to initial resting value (C). Values are means ± SE for mouse cerebral astrocytes (solid lines, n = 3-6) and rat hippocampal astrocytes (dashed lines, n = 4). The mean values for 1 mM NH4Cl include data published previously (Fig. 4 of Ref. 8). Sample responses to each concentration of NH4Cl (mM) are shown on right, arbitrarily displaced on vertical axis for clarity.

After the transient alkalinization, within 5 s of the addition of NH4Cl, the cytosol began to acidify at a rate that increased sharply with increasing NH4Cl concentration and showed no evidence of saturating at up to 20 mM NH4Cl (Fig. 3B). The initial rate of acidification (dpHi/dt) for 1 mM NH4Cl, estimated by linear regression through the first 10 data points after the peak pHi, was 0.589 ± 0.025 pH unit/min (n = 57). This rate of acidification is equivalent to the net entry of H+ at a velocity of 37.2 nmol · min-1 · mg protein-1, calculated using previous estimates of intracellular buffering power (15.8 mmol · l-1 · pH unit-1) and solute accessible intracellular volume (4 µl/mg protein) (10). The acidification can be interpreted as due entirely to the inward transport of NH+4, which dissociates on entry to form NH3. Inward shuttling of protons by such a cycle of NH+4 influx, NH+4 dissociation, and outward diffusion of NH3 was discussed by Boron and De Weer (3). The initial influx of NH+4 should be even faster than indicated by dpHi/dt, because intracellular NH+4 concentration rises as pHi falls and, until a steady-state distribution of ammonia is attained, some of the entering NH+4 does not dissociate to form protons. The maximal acidification induced by 1 mM NH4Cl was 0.366 ± 0.011 pH unit (n = 57), which lowered pHi to 6.77 from an initial resting value of 7.14 ± 0.01 (n = 9) in these experiments.

Figure 3C shows that, in contrast to dpHi/dt, the maximal acidification saturated at >= 5 mM NH4Cl. The amplitude of the acidification may be self-limiting for several reasons. First, the electrochemical driving force for passive NH+4 entry, which approaches the driving force for H+ entry (3), declines as pHi acidifies. Second, the velocity of regulatory acid extrusion is likely to increase at low pHi (36). Third, the conductance of ion channels mediating passive NH+4 transport may decrease as intracellular H+ concentration increases (27).

Comparative responses of rat astrocytes. Figure 3 includes comparative effects of 1-20 mM NH4Cl on pHi in primary rat hippocampal astrocytes. Whereas the amplitude of the transient alkalinization was not different, dpHi/dt and the maximal acidification were much smaller in rat than in mouse astrocytes. For the four concentrations of NH4Cl tested, dpHi/dt in rat astrocytes was a consistent 10.7 ± 0.8% of the rate in mouse astrocytes. This corresponds to the smallest difference in K+ flux observed between mouse and rat astrocytes under various conditions of culture (i.e., 10-fold greater in mouse; see Ref. 46). If it is assumed that these different rates of acidification reflect a difference in NH+4 influx, the lack of a difference in the transient alkalinization (Fig. 3A) is an indication that NH+4 influx was not fast enough, even in mouse astrocytes, to materially reduce the inward gradient of NH3 concentration before its dissipation by NH3 diffusion. All the remaining observations were made with mouse astrocytes.

Effect of the buffer on the response to NH4Cl. Physiological HCO-3 buffering (26 mM HCO-3 in EBS) increases buffering power by ~23 mmol · l-1 · pH unit-1 (12) to a new total of 38.8 mmol · l-1 · pH unit-1. Thus, although dpHi/dt in response to 1 mM NH4Cl declined to 0.203 ± 0.040 pH unit/min (n = 7) in EBS in 5% CO2-95% O2 (Fig. 4) and the maximal acidification declined to 0.107 ± 0.022 pH unit, there was little change in the estimate of the initial rate of H+ entry in the form of NH+4 (31.5 nmol · min-1 · mg protein-1).


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Fig. 4.   Effects of bicarbonate and extracellular pH on response of pHi to 1 mM NH4Cl. A: responses of pHi to 1 mM NH4Cl in bicarbonate-buffered solution [Earle's balanced salts (EBS) in 5% CO2-95% O2], nominally bicarbonate-free solution (HTB in air), and bicarbonate-depleted solution (HTB in 100% O2) are superimposed. B: alkalinization, dpHi/dt, and acidification (as defined in Fig. 3 legend) plotted as a percentage of mean control values (n = 6-8) obtained in HTB in air at pH 7.4. Error bars, SE (n = 5-7). * P < 0.05, different from controls. Initial resting values of pHi (means ± SE, n = 5-7) are shown for each solution.

The observation that in HCO-3-depleted solution (HTB in 100% O2) the acidification induced by 1 mM NH4Cl increased by 73% (Fig. 4) provides further evidence that significant HCO-3-dependent acid extrusion persists, even in nominally HCO-3-free solution (HTB in air), as noted previously (10). The slight reduction in buffering capacity cannot account for this increase. The absence of a significant increase in dpHi/dt when HCO-3-depleted solution was used, similar to the unchanged estimate of NH+4 influx in EBS, is an indication that this rate is relatively insensitive to changes in acid extrusion capacity.

Lowering the pH of the bathing solution to 7.0 decreased the transient alkalinization in response to 1 mM NH4Cl (Fig. 4), whereas raising the pH to 7.8 increased the alkalinization, presumably reflecting the effect of pHo on extracellular NH3 concentration. The rate and the extent of NH4Cl-induced acidification were reduced by lowering pHo to 7.0 but were unaffected by raising pHo to 7.8. The initial resting values of pHi (Fig. 4, bottom) partially followed the changes in pHo, as noted previously (26). Reduced K+ conductance at low pHi and pHo may have inhibited NH+4 influx. However, activation of Na+/H+ exchange by low pHi (18) may also have limited NH+4-induced acidification.

Effect of extracellular [K+] on the response to NH4Cl. Evidence that NH+4 enters cells via K+ transport pathways includes mutual inhibition of transport by these ions. Figure 5 shows the results of experiments examining the effect of extracellular [K+] ([K+]o) on responses of pHi to 1 mM NH4Cl. The reduced rate and amplitude of acidification in the presence of elevated [K+]o are consistent with inhibition of NH+4 entry. K+-induced membrane depolarization will, of course, also affect the conductance of voltage-sensitive ion channels and the driving force for passive NH+4 entry. The intracellular concentration of total ammonia at steady state is expected to decline, because resting pHi increased on raising [K+]o (10). This may account for the decrease in amplitude of the transient alkalinization (Fig. 5A), since less NH3 must enter to reach a steady-state distribution of ammonia.


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Fig. 5.   Response of pHi to NH4Cl depends on extracellular K+ concentration. Plotted against K+ concentration in HTB in air (logarithmic abscissa) are alkalinization (A), dpHi/dt (B), and acidification (C) elicted by 1 mM NH4Cl (as defined in Fig. 3 legend). Error bars, SE; number of coverslip cultures is shown in parentheses. D: sample response to 1 mM NH4Cl at each indicated K+ concentration. Dashed horizontal lines, initial resting pHi (mean ± SE appended). These traces are arbitrarily displaced on vertical axis for clarity.

Contrary to expectation, lowering [K+]o did not increase dpHi/dt and actually reduced the amplitude of the acidification induced by 1 mM NH4Cl (Fig. 5D). Plausible explanations for this result include a modulatory effect of low [K+]o to diminish the conductance of inwardly rectifying K+ (Kir) channels (20). Additionally, the ability of the astrocytes to sustain gradients of K+ and potential is reduced at very low [K+]o and could be further reduced in the presence of NH+4, which might inhibit K+ uptake via the Na+ pump (17). The relatively rapid decline of the NH4Cl-induced acidification with time when [K+]o = 1 mM (Fig. 5D) suggests a reduced ability to sustain the membrane potential. The alkalinizing effect of increasing [K+]o on the initial resting value of pHi (Fig. 5D) reflects the linear relationship between pHi and the logarithm of [K+]o reported previously (10).

Block of NH4Cl-induced acidification by Ba2+. The sensitivity of NH4Cl-induced acidification to inhibition by low concentrations of Ba2+ is shown in Fig. 6. Near-maximal inhibition of the rate and the amplitude of acidification was apparent at 10 µM Ba2+, similar to the Ba2+ sensitivity shown by Kir currents in rat spinal cord astrocytes (34). The initial rate of NH+4-induced acidification decreased by 80.6 ± 1.9% (n = 8) in the presence of 50 µM Ba2+. Ba2+ (10-50 µM) also increased the amplitude of the transient NH4Cl-induced alkalinization by 38-56% (P < 0.05; data not shown).


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Fig. 6.   Low concentrations of Ba2+ inhibit NH+4-induced acidification. A: control response of pHi to 1 mM NH4Cl (dotted trace) is superimposed on response to 1 mM NH4Cl recorded after addition of 10 µM Ba2+ (solid trace). Dashed horizontal line, initial resting pHi. Initial acidification rate (pH units/min; B) and peak acidification (C) elicted by 1 mM NH4Cl are plotted against previously added Ba2+ concentration. Error bars, where they extend beyond symbols, indicate SE (n = 7-11).

In the present study, 10-50 µM Ba2+ did not itself elicit a detectable intracellular alkalinization. However, in previous studies, a higher concentration of Ba2+ (1 mM) alkalinized the cytoplasm as a consequence of plasma membrane depolarization (12). Addition of 5 mM Ba2+ in the present study increased pHi by 0.189 ± 0.006 (n = 2) (see also Ref. 9), yet addition of 1 mM NH4Cl in the continued presence of 5 mM Ba2+ elicited an acidification of 0.14 ± 0.01 pH unit (n = 2), which is comparable to 0.08 ± 0.01 pH unit (n = 8) seen with 50 µM Ba2+. Thus inhibition of NH4Cl-induced acidification is maximal below the Ba2+ concentration range that produces a depolarization-induced alkalinization. This result suggests that depolarization of the membrane and inhibition of NH+4 influx may be associated with block by Ba2+ of different populations of K+ channels. On the other hand, because membrane potential was not measured, a role for membrane depolarization in the inhibition of NH4Cl-induced acidification by 10-50 µM Ba2+ is not ruled out by these data.

Effect of other inhibitors of K+ transport. Bumetanide, a selective inhibitor of the Na+-K+-2Cl- cotransporter, reduced the initial rate and the amplitude of NH+4-induced acidification, but less effectively than Ba2+. The initial acidification rate declined by 33.9 ± 3.1% (n = 6) in the presence of 100 µM bumetanide (Fig. 7C). The combination of 100 µM bumetanide and 50 µM Ba2+ was more effective than Ba2+ alone, preventing any net acidification below the resting pHi in response to 1 mM NH4Cl (Fig. 7A) in each of 10 trials. However, higher concentrations of NH4Cl were able to elicit an acidification in the presence of these combined inhibitors (data not shown), raising the possibility of additional routes of NH+4 entry.


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Fig. 7.   Effects of inhibitors on response of pHi to 1 mM NH4Cl. A: inhibition by 50 µM Ba2+ and 0.1 mM bumetanide (bumet) is cumulative. Traces of responses to 1 mM NH4Cl are superimposed on a dashed horizontal line, indicating initial resting pHi. B: decline of acidification elicited by 1 mM NH4Cl is accelerated by 5 mM ouabain added 2 min previously. C: initial acidification rate and peak acidification (as defined in Fig. 3 legend) plotted as a percentage of mean control values. Error bars, SE [n = 6 for bumetanide and tetraethylammonium (TEA), n = 3 for ouabain]. * P < 0.05, different from controls.

Kir currents in rat spinal cord astrocytes were blocked by 10 mM tetraethylammonium (TEA) and resistant to block by 5 mM 4-aminopyridine (4-AP) (34). However, we found that 10 mM TEA had no effect on the response to 1 mM NH4Cl (Fig. 7C). As a membrane-permeable base, 4-AP itself elicited a large alkalinization (33). This compromised the use of pHi as a probe for ammonia fluxes in the presence of 4-AP.

Because rodent astrocytes express ouabain-resistant Na+-K+-ATPase (41), high concentrations of ouabain (1-5 mM) were used to block this potential pathway for NH+4 entry. Figure 7C shows a significant inhibition of NH4Cl-induced acidification by 5 mM ouabain but no significant reduction in dpHi/dt. More importantly, as illustrated in Fig. 7B, the acidification was not sustained in astrocytes treated with 5 mM ouabain but declined rapidly with time. This suggests that a declining driving force for NH+4 entry by other pathways, rather than block of NH+4 transport by Na+-K+-ATPase itself, may figure prominently in the effect of ouabain.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Our study of the unconventional response of pHi to NH4Cl in mouse astrocytes suggests that the velocity and functional impact of the influx of NH+4 in mammalian astrocytes may be greater than previously supposed. None of our observations challenge the generally held view that diffusion of NH3 is primarily responsible for the rapid distribution of ammonia across the astrocyte plasma membrane (14). However, we interpret our findings to mean that an intense influx of NH+4 persists after the steady-state distribution of ammonia is attained. A driving force for NH+4 entry continues to exist, because the equilibrium potential for NH+4 tends toward the value of the equilibrium potential for H+, which is maintained less negative than the membrane potential of the astrocytes by pH-regulatory transport (36). Once a steady-state distribution of ammonia is reached, all the NH3 formed by dissociation of entering NH+4 during the steady state will diffuse out, and this cycle of NH+4 influx and NH3 efflux imposes a continuing intracellular acid load. From the initial rate of the acidification elicited by 1 mM NH4Cl in bicarbonate-buffered solution, we estimate that NH+4 entered at a velocity of at least 31.5 nmol · min-1 · mg protein-1, which is comparable to the maximal velocity of the avid Na+-dependent uptake of glutamate by these cells (21).

The sensitivity of NH+4-induced acidification to inhibition by low concentrations of Ba2+ (34) strongly suggests that passive influx via Kir channels is the primary entry route for NH+4. Other lines of evidence are consistent with this view. NH+4 is known to permeate K+ channels, and the characteristically high permeability of the astrocyte plasma membrane to K+ at resting potential is generally attributed to Kir conductance (2). The high velocity of NH+4 entry and the apparent lack of saturability of this influx, which are indicated by the observed rates of acidification, are compatible with passive transport. However, regulation of NH+4 transport by pHi (24) potentially could simulate this lack of saturability. Inhibition of NH+4 influx by reduced [K+]o as well as by elevated levels conforms to the observed [K+] sensitivity of Kir conductance (20). The absence of inhibition we found with TEA, although at variance with the susceptibility of Kir conductance seen in spinal cord astrocytes (34), is consistent with the resistance of this conductance to TEA block reported for hippocampal astrocytes (44). Finally, the marked difference in rate of NH+4-induced acidification between mouse and rat astrocyte cultures mirrors the previously reported difference in Ba2+-sensitive K+ flux (45, 46).

Inhibition by bumetanide of one-third of the initial rate of acidification elicited by 1 mM NH+4 suggests that the Na+-K+-2Cl- cotransporter also plays a significant role in mediating NH+4 entry, as reported for other cell types (17, 23). However, it is not clear that the effect of a high concentration of ouabain can be taken to indicate NH+4 transport via Na+-K+-ATPase. The accelerated decline of NH+4-induced acidification in the presence of ouabain is compatible with progressive membrane depolarization and consequent reduction of the driving force for NH+4 influx. The entry of Na+ together with NH+4 via the Na+-K+-2Cl- cotransporter would accelerate such a process. The combined action of Ba2+ and bumetanide eliminated net acidification by 1 mM NH+4. Nevertheless, some influx of NH+4 may have persisted, partially masked by pH-regulatory acid extrusion. Thus additional entry pathways for NH+4 are not excluded by our data. Limits on the specificity of Ba2+ and bumetanide also leave open the possibility of other pathways. For example, a high concentration of Ba2+ was shown to inhibit NH+4 flux via the K+/H+ exchanger in thick ascending limb (1). Also, 100 µM bumetanide partially inhibited K+-Cl- cotransport in red blood cells (19).

The 10-fold lower rate of NH+4-induced acidification in rat astrocytes than in mouse astrocytes more likely reflects a difference in expression of K+ conductance in vitro than a species difference in vivo. The expression of Ba2+-sensitive K+ flux (46), K+-induced alkalinization (33), and Kir conductance in rat astrocyte cultures (15) each was found to be promoted by removal of serum or by treatment with dibutyryl-cAMP. For unknown reasons, the expression of these properties in mouse astrocytes appears less affected by constituents of the nutrient medium (21, 46). Control observations of rat astrocyte cultures in this study were necessitated by earlier reports of a conventional response of rat astrocytes to the ammonia-prepulse technique (7, 26). We did not attempt to confirm the basis for this difference in NH4Cl response. It can be argued that other in vitro models, specifically acutely isolated astrocytes or brain slices, preserve the mature astrocytic phenotype more faithfully than cell culture. However, these models are subject to their own limitations. For example, acutely isolated astrocytes are stripped of fine terminal processes, which may be major sites of K+ conductance (31), during tissue dissociation. In slice preparations the ionic composition of the extracellular compartment cannot be controlled with certainty.


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Fig. 8.   Theoretical relationship between transmembrane distribution ratio of total ammonia and relative membrane permeability to NH3 and NH+4 for 3 different values of pHi. Distribution ratio is given by following expression (see Eq. 3 in Ref. 4)
<FR><NU>[H<SUP>+</SUP>]<SUB>i</SUB> + <IT>K</IT><SUB>a</SUB></NU><DE>[H<SUP>+</SUP>]<SUB>o</SUB> + <IT>K</IT><SUB>a</SUB></DE></FR> ⋅ <FR><NU>(<IT>P</IT><SUB>B</SUB>/<IT>P</IT><SUB>BH<SUP>+</SUP></SUB>) − { <IT>f</IT> [H<SUP>+</SUP>]<SUB>o</SUB>/<IT>K</IT><SUB>a</SUB>(1 − <IT>e</IT><SUP> <IT>f</IT></SUP> )}</NU><DE>(<IT>P</IT><SUB>B</SUB>/<IT>P</IT><SUB>BH<SUP>+</SUP></SUB>) − { <IT>f</IT> [H<SUP>+</SUP>]<SUB>i</SUB><IT>e</IT><SUP> <IT>f</IT></SUP>/<IT>K</IT><SUB>a</SUB>(1 − <IT>e</IT><SUP> <IT>f</IT></SUP> )}</DE></FR>
where [H+]i and [H+]o are intracellular and extracellular H+ concentrations, Ka is association constant, PB/PBH+ is relative permeability to NH3 and NH+4, respectively, and f = FVm/RT (Vm is membrane potential and F, R and T are Faraday's constant, gas constant, and absolute temperature, respectively). Calculations used pHi values shown, extracellular pH = 7.4, pKa = 9.2, Vm = -90 mV, and temperature = 37°C. Inset: expansion of foot of curves.

The buffered solution bathing the cell cultures in this study maintained constant the ionic composition of the extracellular compartment. The extracellular space in vivo is smaller and less well buffered than the intracellular compartment, so extracellular changes can profoundly affect pHi and transmembrane NH+4 fluxes (12). Accordingly, the glial alkalinization observed in experimental models of hyperammonemia can be understood in the context of an associated rise in [K+]o (39). After a 6-h infusion of ammonium acetate that elevated plasma ammonia to 0.63 mM in rats, cerebrocortical [K+]o reached a mean value of 11.8 mM (39). Our data show that high [K+]o and physiological bicarbonate levels markedly attenuate NH+4-induced acidification, whereas K+-induced alkalinization (Fig. 5D) was shown previously to be amplified by physiological bicarbonate buffering (10). The above combination of increased extracellular levels of ammonia and K+ in bicarbonate-buffered solution would induce a net alkalinization in our in vitro astrocyte preparation, consistent with findings in vivo. The intracellular acidification reported in cortical brain slices exposed to 1 mM NH+4 (11) can be attributed to the short diffusion pathway to the superfusion solution limiting accumulation of extracellular K+ in the slice.

The physiological significance of our findings for ammonia distribution in the brain is likely to depend critically on the relative permeability of the astrocytic plasma membrane to the uncharged (B) and charged (BH+) forms of ammonia (PB/PBH+). The importance of PB/PBH+ is illustrated in Fig. 8, which shows the intracellular-to-extracellular distribution ratio of total ammonia plotted as a function of PB/PBH+ according to the expression proposed by Roos and Boron (4, 35). Fluxes of both species are assumed to be passive in this model. The steepness of the relationship derives from the characteristically large resting membrane potential of astrocytes (assigned the value -90 mV in Fig. 8). As Roos (35) noted, his expression approaches the Donnan distribution, dependent on the membrane potential but not on pH, when PB/PBH+ is very small. When PB/PBH+ is very large, as has been assumed previously for astrocytes, the distribution is determined by the transmembrane pH gradient and is independent of membrane potential. A value of PB/PBH+ much below 50-100 (Fig. 8) is unlikely in astrocytes, because the brain-blood distribution of ammonia has been found to range between 1.5 and 3 (14). However, our findings raise the possibility that PB/PBH+ could lie at the foot of the rising phase of the curve. For example, the ammonia distribution ratio is 4.5 when PB/PBH+ = 100 and pHi = 7.2 (a typical value of pHi in bicarbonate-buffered solution; see Fig. 4). This distribution ratio corresponds to a Nernst potential for NH+4 of -40 mV, deviating from the Nernst potential for H+ (approximately -12 mV), but sufficiently removed from the resting membrane potential to provide a substantial driving force for passive NH+4 entry. In the well-studied instance of the thick ascending limb, PB/PBH+ for ammonia is ~20 (16).

The functional implication of relative permeabilities near the center of the range plotted in Fig. 8 is that increasing levels of neuronal activity would modulate astrocytic processing of ammonia from a predominantly "buffering" mode to a predominantly "metabolizing" mode. In quiescent conditions, [K+]o can fall below 3 mM, with a consequent decline of astrocytic pHi <7.2 (10). This would reduce glutamine formation (9) and favor a severalfold accumulation of ammonia in astrocytes (Fig. 8) compared with extracellular and neuronal levels. In contrast, increasing neuronal activity would raise [K+]o and, consequently, 1) increase astrocytic pHi above 7.2 (10), 2) stimulate glutamine synthesis (9), and 3) competitively inhibit NH+4 transport, effectively increasing PB/PBH+. As a result of outcomes 1 and 3, the ammonia distribution ratio would be depressed toward unity (Fig. 8). The inverse changes expected in pHo (12), which are ignored in Fig. 8, would amplify the modulatory effect of [K+]o. This scenario further implies the spatial migration of ammonia from quiescent to active brain regions, a direction opposite to that proposed for K+ (2).

The present findings also suggest that NH+4 influx via active and passive K+ transport pathways could contribute initially to the elevation of [K+]o and astrocyte swelling in acute hyperammonemia. In addition to competing for K+ transport pathways, NH+4 potentially could impede K+ clearance by an effect of NH+4-induced acidification to reduce K+ conductance and gap junctional permeability (38). The electrochemical gradients driving NH+4 entry would also present an osmotic load to the astrocytes in the form of Na+ and Cl- accompanying NH+4 on the Na+-K+-2Cl- cotransporter. There is a documented involvement of Na+-K+-2Cl- cotransport in astrocyte volume regulation (25). Swelling of astrocytic end feet is observed early in hyperammonemia (39).

However, these putative early effects of NH+4 on astrocytic K+ transport in acute hyperammonemia are inherently self-limiting, inasmuch as [K+]o rises and diminishes or reverses them. Other mechanisms, such as the metabolic disturbances addressed by Sugimoto et al. (39), are likely to sustain astrocyte swelling in hyperammonemia.

    ACKNOWLEDGEMENTS

We are indebted to Yvonne Logan for skilled preparation of cultures. The help and advice of Dr. R. James Turner, in whose laboratory the unconventional ammonia response described here was first observed, have been invaluable. Drs. John M. Hamlyn and Bruce P. Hamilton generously provided access to the spectrofluorometer.

    FOOTNOTES

This work was supported by National Institute of Environmental Health Sciences Grant ES-03928 and an Intramural Award from the University of Maryland School of Medicine.

Preliminary reports of this study were communicated previously in brief form (8, 28).

Address for reprint requests: N. Brookes, Dept. of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, MD 21201.

Received 10 June 1997; accepted in final form 21 January 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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