Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21202
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ABSTRACT |
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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 · min1 · 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
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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/HCOCalibration 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
(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
pHi at
a theoretically specified value of
pHi.
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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.
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RESULTS |
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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
HCO3 (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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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
HCO3-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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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 HCO3 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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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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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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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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DISCUSSION |
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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 · min1 · 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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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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