Correspondence to: Païkan Marcaggi, Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK. Fax:0044 171 413 8395 E-mail:p.marcaggi{at}ucl.ac.uk.
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Abstract |
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There appears to be a flux of ammonium (NH4+/NH3) from neurons to glial cells in most nervous tissues. In bee retinal glial cells, NH4+/NH3 uptake is at least partly by chloride-dependant transport of the ionic form NH4+. Transmembrane transport of NH4+ has been described previously on transporters on which NH4+ replaces K+, or, more rarely, Na+ or H+, but no transport system in animal cells has been shown to be selective for NH4+ over these other ions. To see if the NH4+-Cl- cotransporter on bee retinal glial cells is selective for NH4+ over K+ we measured ammonium-induced changes in intracellular pH (pHi) in isolated bundles of glial cells using a fluorescent indicator. These changes in pHi result from transmembrane fluxes not only of NH4+, but also of NH3. To estimate transmembrane fluxes of NH4+, it was necessary to measure several parameters. Intracellular pH buffering power was found to be 12 mM. Regulatory mechanisms tended to restore intracellular [H+] after its displacement with a time constant of 3 min. Membrane permeability to NH3 was 13 µm s-1. A numerical model was used to deduce the NH4+ flux through the transporter that would account for the pHi changes induced by a 30-s application of ammonium. This flux saturated with increasing [NH4+]o; the relation was fitted with a Michaelis-Menten equation with Km 7 mM. The inhibition of NH4+ flux by extracellular K+ appeared to be competitive, with an apparent Ki of ~15 mM. A simple standard model of the transport process satisfactorily described the pHi changes caused by various experimental manipulations when the transporter bound NH4+ with greater affinity than K+. We conclude that this transporter is functionally selective for NH4+ over K+ and that the transporter molecule probably has a greater affinity for NH4+ than for K+.
Key Words: ammonia, K-Cl cotransporter, neuroglia, pH, Apis
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INTRODUCTION |
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Although transmembrane transport of ammonium in animals has been studied, mainly in the mammalian kidney, there are two well-established cases of fluxes of ammonium from neurons to glial cells in nervous tissue. In vertebrate brain, where glutamate is the main neurotransmitter, the uptake of glutamate by astrocytes followed by its amination to glutamine, which is returned to the neurons and deaminated, implies a flux of ammonium (
Uptake of ammonium into cells can be monitored continuously, but indirectly, by measuring the changes in intracellular pH (pHi) that it causes. Ammonium has a pKa of ~9.2 in water (
Several cases have been described of cation-chloride cotransporters, particularly in kidney, being able to transport NH4+ in the place of K+, although with a lower affinity (
Influx of NH4+ into a cell is generally associated with transmembrane fluxes of NH3 (pHi) and NH4+ flux (FNH4)1 is complex. We tackled the question of the NH4+/K+ selectivity in two stages. First, we deduced FNH4 from
pHi for relatively brief applications of ammonium. This required accurate absolute measurements of pHi and measurement of several other parameters: membrane permeability to NH3, intracellular buffering power, and the kinetics of pHi regulation. Use of this "cell model" showed a functional selectivity for NH4+ over K+. We then recorded pHi responses to longer and more complex NH4+ application protocols. By simulating these responses with a standard minimal model for a cotransport process, to which we added competitive inhibition, we estimated the NH4+ and K+ affinities of the transporter molecule.
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MATERIALS AND METHODS |
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Intracellular pH (pHi) in bundles of glial cells dissociated from the retina of the drone (male) Apis mellifera was measured by techniques developed from those described in
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Dissociation Procedure and Loading of the Cells
Bees were obtained from A. Dittlo (Villandraut) or J. Kefuss (Toulouse, France) and maintained on sugar water. A slice of drone head ~500-µm thick was cut with a razor blade. The slice was incubated for 40 min in a 1.5 ml Eppendorf tube containing 1 ml oxygenated Cardinaud solution (see below) to which had been added 2 mg trypsin (T- 4665; Sigma-Aldrich). The slice was washed in Cardinaud solution lacking Ca2+ and Mg2+ and the retinal tissue dissected out and triturated. 150 µl of cell suspension was placed in the perfusion chamber (see below) whose floor consisted of a microscope cover slip coated with poly-L-lysine. The cells were allowed to settle for 10 min and then exposed to the acetoxymethyl ester of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) (Molecular Probes, Inc.) at a concentration of 10 µM for 40 min.
Measurement of Fluorescence
The chamber was placed on the stage of an inverted microscope (Diaphot; Nikon) equipped with a 40x objective, photomultiplier detection, and dual wavelength excitation at 440 and 495 nm switched by liquid crystal shutters, as described in
Solutions
The standard perfusion solution contained (mM): 200 NaCl, 10 KCl, 4 MgCl2, 2 CaCl2. pH was buffered with 10 mM MOPS hemisodium salt and set to 6.90 with HCl. Osmolality was adjusted to 685 mOsm with mannitol (~240 mM). The salt components, the pH, and the osmolarity of this solution are similar to those measured in vivo (
Perfusion System
To be able to make sufficiently rapid solution changes without detaching the cells from the floor of the chamber, we developed a perfusion chamber with no eddy currents. A factor that appeared to be important was the presence of a curved junction between the floor and the wall of the channel (Fig 1 A). Solutions were gravity fed and selected by computer-controlled solenoid valves whose outflows passed through fine tubes at ~30 µl s-1 into a common pathway to the chamber. It was found that mixing of solutions was negligible. We obtained a measure of the speed of the solution change in the chamber by recording the change in fluorescence during a switch from standard solution to one containing 1 µg liter-1 fluorescein (Fig 1 B). The change in pHi measured with BCECF in response to propionate or trimethylamine (TMA) was nearly as fast (see Fig 4 B and 5 A). The change in fluorescein fluorescence was well described by an exponential; for flow rates used in experiments with cells, the mean time constant was: 5.4 ± 1.9 s (± SD, n = 16), and this exponential was used to describe the changes in extracellular concentration in our numerical models.
Calibration of pHi Measurements
We initially used two techniques for calibrating pHi. To estimate the shape of the curve that gives pHi as a function of I440/I495, the cell membranes were made permeable to H+ with nigericin so that pHi varied with pHo (
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(1) |
The values obtained for the constants were 6.93 ± 0.03 for pK and 0.991 ± 0.022 for b (n = 11). The advantage of this procedure is that calibration for each experiment is reduced to obtaining the fluorescence ratio corresponding to pHi 6.84. This ratio was obtained by superfusing the cells with 2 mM NH4+ at pHo 6.90, a procedure that we found to give a pHi 6.84 (see Fig 3, AD).
Absolute Measurement of pHi
The null method of apHi be the change of pHi that would have been produced by superfusion with a concentration aC of a weak acid AH + A- and
bpHi that for a concentration bC of a weak base BH+ + B. Assuming that the diffusion of the neutral form (AH or B) and its re-equilibration with the charged form in the cell are rapid compared with pHi regulatory mechanisms, then:
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(2a) |
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(2b) |
where ßi is the buffering power. Let pHi be the net pHi change produced by a simultaneous application of a concentration aC of AH + A- and bC of BH+ + B.
pHi =
apHi +
bpHi.
When pHi
0, it follows from Equation 2a and Equation 2b that:
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(3) |
To determine the ratio bC/aC for which pHi
0, two pairs of concentrations (aC; bC1) and (aC; bC2), which gave rise to
pHi1 and
pHi2 were applied successively. The desired bC was then estimated from:
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(4) |
This method is most accurate when bC/aC = 1 and hence when pHi = pHo; we were able to bring pHi close to pHo by applying NH4+ (see Fig 3, AD).
Comparison of the Permeabilities of the Neutral Forms of a Weak Base and a Weak Acid
We choose a weak acid AH/A- whose pKa = apKa < 5 so that at pHo [6; 8] its total concentration Ca = [AH]o +[A-]o
[A-]o. We choose a weak base BH+/B whose pKa = bpKa > 9 so that at pHo
[6; 8] its total concentration Cb = [BH+]o + [B]o
[BH+]o. We set Ca = Cb and find the pHo (
[6; 8]) for which the initial inward transmembrane flux of B (FB = PB x [B]o) is equal to that of AH (FAH = PAH x [AH]o):
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(5) |
For pHi [6; 8], the initial rate of pHi change induced by the weak acid is -FAH/ßi since, in the cell, most of AH dissociates to form A- + H+; similarly, the initial rate of pHi change induced by the weak base is FB/ßi. Thus, if one of the permeabilities is known, the other permeability can be deduced from the value of pHo for which the initial direction of the pHi change during the application of the mixture of the weak base and the weak acid reverses (FB = FAH).
Online Supplemental Material
The arguments leading from the observed changes in pHi to the properties of the transporter molecule involve a model of transmembrane fluxes in the cell (essentially that used by
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RESULTS |
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In agreement with
The Ammonium-induced Decrease in pHi Is Inhibited by a High Concentration of K+o
Fig 2 A illustrates how 2 mM ammonium applied for 30 s to an isolated bundle of bee retinal glial cells at the measured physiological pHo of 6.90 (
Parameters to be Determined
Fig 2 B shows a response to a longer (5 min) application of ammonium on an expanded time scale. This response can be divided into five phases that can be explained by the schemes of Fig 2 C (see also NH4+ is so far to the right at pH near 7, it is sufficient that the inward flux of NH3 exceeds ~1% of the inward flux of NH4+. As the ratio [NH4+]i/NH3]i
[H+]i, Phase 1 is expected to be greater for cells with acid pHi. This was actually the case: Phase 1 was detected only for cells with baseline pHi < 7.2. When the NH3 concentrations approach equality on each side of the cell membrane, there is still an inward NH4+ gradient because [H+]o > [H+]i. Then the NH3 flux becomes outward while NH4+ continues to enter the cells and release H+ ions (Phase 2). A steady state is reached (Phase 3) when the production of H+ ions equals their extrusion by pH regulatory processes. When extracellular ammonium is suddenly removed, intracellular ammonium exits the cell faster in the NH3 form than in the NH4+ form, so NH4+ dissociates to form NH3 and there is a rebound acidification (Phase 4), followed by a slower return to baseline as proton equivalents are pumped out of the cell (Phase 5).
reg), and the Cl- concentration gradient that can help drive NH4+ into the cell.
Absolute Determination of pHi During Application of NH4+
The precise value of pHo - pHi in the presence of external ammonium (the plateau phase) is related to the force driving NH4+ across the membrane and is our main motivation for seeking an accurate measure of pHi. pHi (10 mM propionate, 10 mM TMA) and
pHi (10 mM propionate, 5 mM TMA) were in opposite directions, and we estimated by linear interpolation the concentration of TMA that would have given no change in pHi when applied with 10 mM propionate (Equation 4). The absolute value of pHi was then calculated by Equation 3. The method assumes that intracellular pKa equals extracellular pKa and that the membranes are relatively impermeable to the charged forms of the weak acid and base. This latter assumption was confirmed by the observation that, during applications of propionate (n = 21; not shown) or TMA (see Fig 5 A), recovery of pHi was slow and could be fully accounted for by pH regulatory processes. Since pHi during the plateau phase depends partly on pHi regulatory processes (Fig 2 C 3), short NH4+ applications at the beginning and end of the experiment were made to check that the rates of recovery remained approximately the same. From 12 experiments, as in Fig 3 A, pHi was calculated to be 6.844 ± 0.017 (±SD, n = 12) after an 8-min application of NH4+ with pHo = 6.90. This pHi was significantly less than pHo (P < 0.0001) and remained so for at least 35 min (Fig 3 B). To see whether the result depended on the specific weak acid and weak base used, we used other weak acid/weak base couples. In experiments similar to that of Fig 3 A, the estimated difference pHo - pHi after >10 min of perfusion with 2 mM ammonium was not significantly different when the following couples were used: propionate/TMA; propionate/MA; acetate/TMA and caproate/TMA (Fig 3 C). To see whether the value of (pHo - pHi) reached during the plateau phase was related to the baseline pHi, we compared cells with a baseline pHi < 7.1 with those with pHi > 7.1 (Fig 3 C, first two columns). The difference in the mean values of (pHo - pHi) during the plateau phase was not significant. In contrast, as illustrated in Fig 3 A, the level of the plateau did indeed depend strongly on the pHo at which the NH4+ was applied. Absolute values of pHi estimated during superfusion with 2 mM NH4+ at pHo 6.500 ± 0.005, 6.900 ± 0.005, 7.300 ± 0.005, and 7.700 ± 0.005 are plotted in Fig 3 D and show a very precise linear correlation with pHo such that (pHi - 6.142) = 0.9264 x (pHo - 6.142). In later experiments, we calibrated the measurements of pHi simply by superfusing the cells with 2 mM ammonium for at least 8 min and using this relation.
Intracellular Buffering Power
The H+ ions released into (or taken up from) the cytoplasm as a consequence of the transmembrane fluxes of NH4+ and NH3 affect pHi according to the relation pHi =
Q/ßi, where
Q is the quantity of H+ ions/U volume and ßi is the intracellular buffering power (see
To see if ßi varied markedly with pHi, we shifted pHi by applying NH4+ at various pHos. The results and the analysis, which is complicated by the effects of the NH3/NH4+ system, are given in
Permeability to NH3
We estimated NH3 permeability (PNH3) from measurements of pHi under conditions in which entry of NH4+ was blocked so that changes in pHi were due only to the inward flux of NH3. We have previously shown that NH4+ does not enter through barium-sensitive K+ channels, the major cationic conductance in these cells, and also that NH4+ entry is totally blocked by bumetanide or by removal of external chloride (
With the cell bundles adhering to the floor of the perfusion chamber, we failed to find a molecule causing a 1090% pHi change faster than the one produced by ammonium: perhaps the change of solution at the cell membrane (020 µm from the floor of the chamber) was not fast enough for this measurement. To expose cells to faster solution changes, we caught hold of bundles of cells with a 3-µm tip diameter pipette and carried them 50100 µm up from the floor of the chamber. To increase the time resolution of the rapid initial slope of the pH change, we measured the fluorescence ratio with faster switching of the excitation wavelengths (>3 Hz). To reduce delays due to diffusion, we applied ammonium at a high concentration (10 mM) but at an acid pH (6.50) so that [NH3]o was low but benefited from facilitated diffusion (pHi, baseline pHi was reduced (to ~6.80) by perfusing the cells for 3060 min with solution buffered at pHo 6.20. After a 1-min perfusion with 0 Cl- + 0.5 mM bumetanide, 10 mM ammonium was applied at pHo 6.50 (Fig 4 A). In these conditions, the 1090% pHi change induced by 10 mM propionate (Fig 4 B) was twice as fast as the one induced by ammonium, showing that the speed of solution change at the cell membrane did not significantly limit the influx of NH3.
Although pHi was ~7.00 during the ammonium application, while pHo was 6.50, no slow pHi decrease was observed, as would have been the case if the membranes had had some permeability to NH4+. This confirms that NH4+ pathways were insignificant in these conditions.
The slope of the pHi change was measured at 50% of the pHi response, where it is known that [NH3]o has reached its final concentration, since the effect of propionate is 90% at this time (Fig 4). Since, for pHi ~ 7.00, [NH4+]i > 100 x [NH3]i, then ßi x
pHi/
t =
[NH4+]i/
t
([NH4+]i + [NH3]i)/
t = FNH3 x S/V, where FNH3 is the NH3 transmembrane flux and S/V is the ratio of membrane surface to intracellular volume in which the ammonium is distributed. The ratio of the surface to the total cell volume has been estimated to be
1.2 µm-1 (
V/S x ßi x
pHi/
t. From this flux, we found PNH3 = 14.7 ± 2.9 µm · s-1 (n = 6) in 0 Cl- + 0.5 mM bumetanide, which was not significantly different from the value in 0 Cl- only (n = 5), showing that bumetanide did not further inhibit NH4+ entry.
Holding up the cells with a pipette will have introduced some stress in the cell membrane, which may have modified its permeability. To check that NH3 permeability is the same for cells plated on the bottom of the chamber (the conditions used for the other experiments), we also determined PNH3 by an indirect method. Methylamine (MA; CH3NH3+/CH3NH2) is a weak base with a pKa that is high (10.6;
9.2). Because of this pKa difference, at pH < 8 (at which charged forms are preponderant), if [CH3NH3+] + [CH3NH2] = [NH4+] + [NH3], then [CH3NH2] < 0.04 x [NH3]. It follows that if PCH3NH2 (PMA) is not far different from PNH3, for equal concentrations of MA and ammonium applied, FCH3NH2 << FNH3. This is why PMA can be measured directly even with a slow speed of solution change at the cell membrane. Fig 5 A illustrates the pHi response to 10 mM MA compared with the pHi response to 10 mM TMA (pKa
9.6). The speed of the pHi change induced by TMA was far faster than the one induced by MA, showing that the speed of the solution change was fast enough for measurement of PMA, which was found to be 27.4 ± 8.1 µm s-1 (n = 8). Once this permeability was known, it was possible to deduce the permeability of propionate by ascertaining the initial direction of the pHi change induced by a simultaneous application of 10 mM propionate and 10 mM MA. To avoid too great a variation of the net
pHi during this simultaneous application, we used a condition in which pHi
pHo, which was obtained by including 2 mM ammonium in the superfusate (Fig 3 D). As illustrated in Fig 5 B, the initial direction of the pHi change reversed for 7.40 < pHo < 7.60 (n = 4). Taking the mean of this range (pHo
7.50 ± 0.10) gives x = PMA/Pprop
3.16 ± 1.46 according to Equation 5. So, Pprop = PMA/x
8.67 ± 6.57 µm s-1.
The same protocol was used to estimate PNH3 from the now known Pprop, the simultaneous application being done in 0 Cl- to prevent the entry of NH4+. As illustrated in Fig 5 C, the initial direction of the pHi change reversed for 7.00 < pHo < 7.10 (n = 4), which gives x = PNH3/Pprop 1.02 ± 0.24 according to Equation 5. So PNH3 = x x Pprop
8.84 ± 8.78 µm s-1.
In conclusion, the two methods of estimation of PNH3 gave values not significantly different. The standard deviation obtained with the second method was increased by the successive approximations so we give more weight to the value obtained with the first method and conclude that PNH3 is in the range of 719 µm s-1; we take the value 13 µm s-1 for the model.
pH Regulation
When pHi falls below its baseline value, pH regulatory mechanisms tend to restore it by extruding H+ ions. To quantify the kinetics of this regulation, we acid loaded the cells by exposure to ammonium and analyzed the recovery (), where [H+]
was the baseline [H+]i at rest. Linear regressions showed that the recoveries were exponential irrespective of the initial displacement, and had slopes (= -1/
reg) that were not systematically different.
Values of the time constant reg for 17 bundles of cells for which at least three different ammonium concentrations were tested were plotted as a function of the initial pHi displacement,
pHi(NH4+) (Fig 6 B). Linear regression of
reg[
pHi(NH4+)] confirmed that
reg was independent of the pH [mean slope of 0.3 ± 1.8 min (pH unit)-1; n = 17]. We conclude that despite considerable variability,
reg was approximately constant irrespective of the initial pHi displacement with a mean value of 3.0 ± 1.1 min (n = 17). We therefore described the pHi regulation by Equation 6:
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(6) |
with reg = 3 min.
Driving Force
The flux rate of a Cl- cotransporter will depend in part on [Cl-]o and [Cl-]i, and we will use values of these concentrations in the transporter model of Fig 10 A (see online supplemental material). [Cl-]o being known, we attempted to estimate [Cl-]i. Measurements in slices of bee retina with ion-selective microelectrodes have shown that in the glial cells Cl- (and also K+) are at close to electrochemical equilibrium (
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Concentration Dependence of the pHi Changes Induced by 30-s Applications of Ammonium
To record the responses to increasing concentrations of NH4+ in the absence of external K+, we superfused the cells in 0 K+ for 15 s before and during each NH4+ application (Fig 7 A). Repeated exposure to high [NH4+] appeared to lead to impairment of pHi regulation and, for 7 of 11 experiments, pHi did not recover from the acidification induced by 10 mM NH4+. Measurements were therefore made only on the records from the four experiments for which pHi recovered from 10 mM NH4+ and for which the response to subsequent control application of 0.5 or 1 mM NH4+ was closely similar to the initial response (in the record of Fig 7 A, a final application of 20 mM NH4+ was made). To make sure that the effect of NH4+ was not rate limited by the speed of the solution change (as was probably the case in the previous study by
Fig 7 B shows the NH4+ responses from the record of Fig 7 A on a shorter time scale. The time of onset of the response to propionate (not shown) indicated that in this experiment there was a dead time of ~5 s between the switching of the electromagnetic valves and the arrival of a new solution at the cell membrane. The slope of the NH4+-induced pHi change (pHi/
t) was measured before the rebound (Phase 4), between 15 and 35 s after the valves were actuated.
pHi/
t(NH4+) was calculated by linear regression as shown in Fig 7 C.
pHi/
t(NH4+) increased with [NH4+]o in the range 0.510 mM NH4+; but for 20 mM NH4+, although the total pHi change induced by NH4+ [
pHi(NH4+)] continued in every case to increase, in three of the four experiments,
pHi/
t for 20 mM NH4+ was less than for 10 mM, as in the example shown in Fig 7AC. Mean data from the four experiments are shown in Fig 7 D.
pHi([NH4+]o) was well fitted by a Michaelis-Menten curve
pHi([NH4+]o) = a[NH4+]o/(b + [NH4+]o) (R = 0.993) with half saturation, b, for [NH4+]o = 6.62 ± 0.57 mM (n = 4). But
pHi/
t([NH4+]o) could only be fitted by a Michaelis-Menten curve for [NH4+]
5 mM (R = 0.926), half saturation being at [NH4+]o = 3.37 ± 0.98 mM (n = 6) (Fig 7 D). Because the relation between NH4+ transport and pHi changes is indirect, the value of [NH4+]o that half saturates pHi changes does not necessarily correspond to the one that half saturates transport of NH4+. To deduce the flux of NH4+, we had recourse to a mathematical model.
Dependence of NH4+ Flux on [NH4+]o
Three transmembrane fluxes determine pHi during and after application of ammonium (Fig 2 C). Of these, we have a phenomenological description of the pHi regulation (Freg in Fig 2 D), and we assume that the flux of NH3 (FNH3 in Fig 2 D) results from simple diffusion (Fick's law). To deduce the flux of NH4+ through the cotransporter (FNH4 in Fig 2 D) from the changes in pHi, we use the model of Fig 2 D, expressed mathematically in the supplemental material. From the measurements described above, values for parameters of the model were: ßi = 12 mM, pHi = 7.4, reg = 3 min, and PNH3 = 13 µm s-1. The surface-to-volume ratio, S/V, with its attendant uncertainty, was used to calculate PNH3, but cancels out in the calculations.
As a first step, a constant inward FNH4 (inFNH4) was imposed for 30 s, with [NH4+]o (+ [NH3]o) set to 2 mM. The resulting pHi/
t was calculated 15 s after the onset of the imposed inFNH4 and plotted against inFNH4 for various PNH3 (7, 13, and 19 µm s-1; Fig 8 A). Increasing PNH3 increased
pHi/
t, but only slightly, showing that PNH3 is not a major rate-limiting factor.
A similar simulation, still using an imposed inFNH4, was then performed in the presence of various [NH4+]o (+ [NH3]o). Increasing [NH4+]o increased [NH3]o, reduced outward FNH3, and, as expected, reduced pHi/
t(inFNH4) (Fig 8 B). It is clear that the experimental result in which
pHi/
t was smaller for an application of 20 mM ammonium than for 10 mM (Fig 7) does not necessarily imply that inward FNH4(20 mM NH4+) < inward FNH4(10 mM NH4+). We also note that since the relation of
pHi/
t to inFNH4 is curved (Fig 8A and Fig B),
pHi/
t vs. [NH4+]o will saturate more rapidly than will inFNH4 vs. [NH4+]o.
The pHi peak reached after withdrawal of external ammonium must depend both on [NH4+]i at the end of the ammonium application, and on the effluxes of NH4+ and of NH3 after withdrawal. To start modeling this, we considered the case of a 30-s application of 2 mM extracellular NH4+ with a constant inFNH4 (6.65 mM min-1 in Fig 8 C). After removal of extracellular NH4+, the concentration gradient of NH4+ is outwards. We tested the simplest reasonable assumption, which is that outward FNH4 = outFNH4 [NH4+]i. With no loss of generality, this can be written: outFNH4 = outFNH4 max x ([NH4+]i/[NH4+]i max), where [NH4+]i max is the intracellular NH4+ concentration reached at t = 30 s and outFNH4 max is an initially arbitrary constant corresponding to the maximum transient outFNH4. As illustrated in Fig 8 C, the rebound acidification on removal of extracellular NH4+ is maximal for zero outFNH4 and decreases with increasing outFNH4. Let
pHi(inFNH4) be the total pHi change induced by a 30-s inFNH4 followed by an outFNH4 defined as above. From the experimental data, the mean ratio
pHi/(
pHi/
t) measured from 30-s applications of 2 mM NH4+ in 0 K+o was 0.60 ± 0.12 min (n = 10); the closest approach to this in Fig 8 C is 0.56 min for outFNH4 max = 0, which we accept as an approximation.
pHi(inFNH4) is very little affected by [NH4+]o (still for an imposed inFNH4; Fig 8 D), much less so than is
pHi/
t (Fig 8 B). Thus, the inverse operation of estimating inward FNH4([NH4+]o) is better done from
pHi([NH4+]o) than from
pHi/
t([NH4+]o). By comparing experimental
pHi([NH4+]o) (Fig 7 D) and simulated
pHi(inFNH4) (Fig 8 D), we calculated inFNH4([NH4+]o) (Fig 8 E). The points were well fitted by a Michaelis-Menten equation of the form (Equation 7):
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(7) |
The constant, K 'm, corresponding to half saturation of inward FNH4, was 7.8 ± 0.7 mM. Variant analyses {from pHi/
t([NH4+]o or using Lineweaver-Burke plots} all gave lower values, down to 4.9 m
Functional Selectivity for NH4+ over K+
Having established the dependence of inFNH4 on [NH4+]o (for 30-s applications of ammonium), we then extended the approach to analyze the inhibitory effect of K+. Fig 9 A shows an experiment in which cells were superfused for 30 s with NH4+ in 0 or 10 mM K+. pHi([NH4+]) from six such experiments is shown plotted with double inverse scales as a function of [NH4+]o in Fig 9 B. Using the model, as described above, a value of inFNH4([NH4+]) was deduced for each measurement of
pHi([NH4+]) and a second inverse plot was made (Fig 9 C). This plot suggests that the inhibition was competitive since straight lines passing through the data points intersect near the ordinate axis (same inFNH4 max).
Fig 9 D illustrates how K+ depolarizes these glial cells. In this record, from a glial cell in a retinal slice, the depolarization is greatly damped by electrical coupling between the cells and the slowness of the increase in [K+] in the extracellular clefts (pHi(NH4+) in the absence of changes in membrane potential. In confirmation of
pHi(NH4+) (n = 5; not shown). Nor did it have a significant effect on the inhibition of
pHi(NH4+) produced by raising K+ to 20 mM (n = 11; not shown). Hence, the depolarization is unlikely to be responsible for the inhibition of NH4+ transport by extracellular K+.
To quantify the inhibitory effect of K+, we calculated an apparent inhibitory constant, K 'i, defined by: K ''m = K 'm (1 + [K+]o/K 'i), where K 'm is the Michaelis-Menten constant estimated above from responses to NH4+ in 0 K+ and K ''m is the constant estimated from the responses to NH4+ in 10 mM K+. K 'i was found to be 26.7 mM, which is greater than K 'm (7.8 mM). Variant analyses also gave K 'i > K 'm (see
Affinities of the Transporter Molecule for NH4+ and K+
In the previous two sections, we established the dependence of the mean inward FNH4 on [NH4+]o during 30-s (brief) applications of ammonium, and the inhibition of this flux by [K+]o. We now describe the changes in pHi under more varied conditions; notably, longer applications of NH4+. These more complex responses impose additional constraints on the interpretation of the underlying processes and allow us to test whether the transport can be described by a standard minimal kinetic model of membrane cotransport to which we add competition by K+ for the NH4+ binding site (Fig 10 A). As explained by
In the experiment of Fig 10 B, [NH4+]o was increased in steps, each lasting 8 min. The level of the plateau phase (Phase 3 in Fig 2 B) rose no further for [NH4+]o > 5 mM (n = 4). Fig 10 C shows simulated responses to the same protocol of stepwise increases in [NH4+]o for a cell containing the transporter model of A with Ki = 15 mM and Km = 5 mM (continuous trace) or 20 mM (dashed trace). It is seen that the time course of pHi, particularly for the step change [NH4+]o from 5 to 10 mM, is better simulated with Km = 5 mM; i.e., with Km < Ki.
Increasing [K+]o in the Presence of NH4+
Fig 11 A illustrates the inhibitory effect on NH4+ transport of increasing [K+]o during the plateau phase induced by a long application of NH4+. An increase in [K+]o from 10 to 50 mM rapidly increased pHi by 0.092 ± 0.012 pH unit in 2 min in 2 mM NH4+ (n = 5) and by a greater amount, 0.115 ± 0.022 pH unit in 20 mM NH4+ (n = 5). The difference is significant with P = 0.01. This observation raised the question of whether the inhibition by [K+]o was purely competitive.
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Simulations were performed with the transporter model of Fig 10 A and the protocol of Fig 11 A. Km was set to 7 mM. Simulations with Ki = 10, 15, and 20 mM (Fig 11 B) show that inhibition by a 2-min increase in [K+]o from 10 to 50 mM differed from the experimental record in three aspects. First, the inhibition in 20 mM NH4+, although larger than the inhibition in 2 mM NH4+, was not as markedly larger as in the experiments. Second, the increases in pHi induced by rises in [K+]o were slower than the experimental ones. Third, after returning to 10 mM K+, the small rebound acidification present in the experimental records was not reproduced. A transporter model in which inhibition by extracellular K+ was noncompetitive (Fig 11 C, legend) corrected these failings, but excessively so. We did not attempt to fit the experimental data more precisely since our transporter model is highly simplified, but these comparisons to simulations do suggest that inhibition by extracellular K+ may be partly noncompetitive.
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DISCUSSION |
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Sensitivity to loop diuretics and external chloride (
Parameters for the Cell Model: pHi, ßi, PNH3
The null method of pH measurement used on the isolated bundles of glial cells showed that many bundles had pHis at least as alkaline as those measured with pH microelectrodes in slices of retina (mean: 7.31;
Membrane Potential, Cl- Gradient, and pH Regulation
A major difference, potentially important for certain cell functions, between the glial cells in the isolated bundles and those on which published results were obtained in slices of bee retina, is the apparent membrane potential. On the assumption that application of nigericin caused H+ to distribute across the membrane with the same passive distribution as K+, we concluded that mean Vm in the bundles was -4 mV. Support for a small Vm is given by the observation (
Transporters of the cation-Cl- family are normally electroneutral, and the effect of ammonium on glial cell Vm in bee retinal slices is compatible with electroneutral transport (
NH4+/K+ Selectivity of the Transporter
Until now, the few studies of competition between K+ and NH4+ for inward transport into animal cells on transporters have reported a selectivity for K+ (
Reported values for Km (K+) calculated for K+ influx by Cl--dependent transport into erythrocytes are 55 mM (sheep;
Possible Advantages of Glial Uptake of Ammonium in the NH4+ Form
We have shown that ammonium enters bee retinal glial cells overwhelmingly in the NH4+ form. It is striking that this is also the case for mammalian astrocytes (at least those cultured from neonatal mice), although, in contrast to the bee glial cells, the NH4+ entry into cultured astrocytes appears to occur mainly through Ba2+-sensitive channels (
A major ammonium-consuming process in bee retinal glial cells is the conversion of pyruvate to alanine (
In the case of bee retinal glial cells, the ammonium consumption can be summarized by the reaction: CH3-CO-COO- + NH4+ + NADH + H+ CH3-CHNH3+-COO- + H2O + NAD+.
Since this reaction consumes H+, pHi is better conserved if ammonium is supplied in the NH4+ form. In astrocytes, the pathways of energy metabolism are still a matter of debate (see, e.g.,
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Footnotes |
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Dr. Marcaggi's present address is Department of Physiology, University College London, London WC1E 6BT, UK.
The online version of this article contains supplemental material.
1 Abbreviations used in this paper: BCECF-AM, acetoxymethyl ester of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein); FNH4 (and FNH3), transmembrane fluxes of NH4+ (and NH3) per liter of cell; TMA, trimethylamine.
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Acknowledgements |
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We thank Dr. Robert Dantzer for laboratory facilities and Jean-Louis Lavie for his contribution to the set up.
Financial support was received from the Conseil Régional d'Aquitaine (97-0301208).
Submitted: 5 November 1999
Revised: 10 May 2000
Accepted: 11 May 2000
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References |
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Antonenko, Y.N., Pohl, P., Denisov, G.A. 1997. Permeation of ammonia across bilayer lipid membranes studied by ammonium ion selective microelectrodes. Biophys. J. 72:2187-2195[Abstract].
Benjamin, A.M., Quastel, J.H. 1975. Metabolism of amino acids and ammonia in rat brain cortex slices in vitro: a possible role of ammonia in brain function. J. Neurochem. 25:197-206[Medline].
Bertrand, D. 1974. Etude des propriétés électrophysiologiques des cellules pigmentaires de la rétine du faux-bourdon (Apis mellifera). Thesis No. 1650. Université de Genéve.
Boron, W.F., De Weer, P. 1976. Intracellular pH transients in squid axons caused by CO2, NH3, and metabolic inhibitors. J. Gen. Physiol. 67:91-112[Abstract].
Boyarsky, G., Ganz, M.B., Sterzel, R.B., Boron, W.F. 1988. pH regulation in single glomerular mesangial cells I. Acid extrusion in absence and presence of HCO3-. Am. J. Physiol. Cell Physiol. 255:C844-C856
Boyarsky, G., Hanssen, C., Clyne, L.A. 1996. Inadequacy of high K+/nigericin for calibrating BCECF. Am. J. Physiol. Cell Physiol. 271:C1131-C1156
Cardinaud, B., Coles, J.A., Perrottet, P., Spencer, A.J., Osborne, M.P., Tsacopoulos, M. 1994. The composition of the interstitial fluid in the retina of the honeybee drone: implications for the supply of substrates of energy metabolism from blood to neurons. Proc. R. Soc. Lond. B Biol. Sci. 257:49-58.
Chang, A., Hammond, T.G., Sun, T.T., Zeidel, M.L. 1994. Permeability properties of the mammalian bladder apical membrane. Am. J. Physiol. Cell Physiol. 267:C1483-C1492
Coles, J.A., Marcaggi, P., Lavie, J.L. 1999. A rapid wavelength changer based on liquid crystal shutters for use in ratiometric microspectrophotometry. Pflügers Arch. 437:986-989.
Coles, J.A., Marcaggi, P., Véga, C., Cotillon, N. 1996. Effects of photoreceptor metabolism on interstitial and glial cell pH in bee retina: evidence for a role for NH4+. J. Physiol. 495:305-318[Abstract].
Coles, J.A., Orkand, R.K. 1983. Modification of potassium movement through the retina of the drone (Apis mellifera male) by glial uptake. J. Physiol. 340:157-174[Abstract].
Coles, J.A., Orkand, R.K., Yamate, C.L. 1989. Chloride enters glial cells and photoreceptors in response to light stimulation in the retina of the honey bee drone. Glia. 2:287-297[Medline].
Coles, J.A., Orkand, R.K., Yamate, C.L., Tsacopoulos, M. 1986. Free concentrations of Na, K and Cl in the retina of the honeybee drone: stimulus-induced redistribution and homeostasis. Ann. NY Acad. Sci. 481:303-317[Medline].
Coles, J.A., Rick, R. 1985. An electron microprobe analysis of photoreceptors and outer pigment cells in the retina of the honey bee drone. J. Comp. Physiol 156:213-222.
Cougnon, M., Bouyer, P., Jaisser, F., Edelman, A., Planelles, G. 1999. Ammonium transport by the colonic H+-K+-ATPase expressed in Xenopus oocytes. Am. J. Physiol. Cell Physiol. 277:C280-C287
Deitmer, J.W., Rose, C.R. 1996. pH regulation and proton signalling by glial cells. Prog. Neurobiol. 48:73-103[Medline].
Delpire, E., Lauf, P.K. 1991. Kinetics of Cl-dependant K fluxes in hyposmotically swollen low K sheep erythrocytes. J. Gen. Physiol. 97:173-193[Abstract].
Demestre, M., Boutelle, M.G., Fillenz, M. 1997. Stimulated release of lactate in freely moving rats is dependent on the uptake of glutamate. J. Physiol. 499:825-832[Abstract].
Eisner, D.A., Kenning, N.A., O'Neill, S.C., Pocock, G., Richards, C.D., Valdeolmillos, M. 1989. A novel method for absolute calibration of intracellular pH indicators. Pflügers Arch. 413:553-558.
Engasser, J.M., Horvath, C. 1974. Buffer-facilitated proton transport pH profile of bound enzymes. Biochim. Biophys. Acta. 358:178-192[Medline].
Evans, R.L., Turner, R.J. 1998. Evidence for physiological role of NH4+ transport on secretory Na+-K+-2Cl- cotransporter. Biochem. Biophys. Res. Commun. 245:301-306[Medline].
Good, D.W. 1994. Ammonium transport by the thick ascending limb of Henle's loop. Annu. Rev. Physiol. 56:623-647[Medline].
Haas, M., Forbush, B.R. 1998. The Na-K-Cl cotransporters. J. Bioenerg. Biomembr. 30:161-172[Medline].
Hassel, B., Bachelard, H., Jones, P., Fonnum, F., Sonnewald, U. 1997. Trafficking of amino acids between neurons and glia in vivo. Effects of inhibition of glial metabolism by fluoroacetate. J. Cerebr. Blood Flow Metab. 17:1230-1238[Medline].
Hille, B. 1992. Ionic Channels of Excitable Membranes. 2nd ed Sunderland, MA, Sinauer Associates, Inc, pp. 607 pp.
Jacobs, M.H. 1940. Some aspects of cell permeability to weak electrolytes. Cold Spring Harbor Symp. Quant. Biol. 8:30-39.
Kaiser, B.N., Finnegan, P.M., Whitehead, L.F., Bergersen, F.J., Day, D.A., Udvardi, M.K. 1998. Characterization of an ammonium transport protein from the peribacteroid membrane of soybean nodules. Science. 281:1202-1206
Kaji, D. 1989. Kinetics of volume-sensitive K transport in human erythrocytes: evidence for asymmetry. Am. J. Physiol. Cell Physiol. 256:C1214-C1223
Kenyon, J.L., Gibbons, W. R. 1977. Effects of low chloride solutions on action potentials of sheep cardiac purkinje fibers. J. Gen. Physiol. 70:635-660
Kinne, R., Kinne-Saffran, E., Schütz, H., Scholermann, B. 1986. Ammonium transport in medullary thick ascending limb of rabbit kidney: involvement of the Na+, K+, Cl--cotransporter. J. Membr. Biol. 94:279-284[Medline].
Klocke, R.A., Andersson, K.K., Rotman, H.H., Forster, R.E. 1972. Permeability of human erythrocytes to ammonia and weak acids. Am. J. Physiol. 222:1004-1013[Medline].
Labotka, R.J., Lundberg, P., Kuchel, P.W. 1995. Ammonia permeability of erythrocyte membrane studied by 14N and 15N saturation transfer NMR spectroscopy. Am. J. Physiol. Cell Physiol. 268:C686-C699
Lowry, O.H., Passoneau, J.V. 1966. Kinetic evidence for multiple binding sites on phosphofructokinase. J. Biol. Chem. 241:2268-2279
Magistretti, P.J., Pellerin, L., Rothman, D.L., Shulman, R.G. 1999. Energy on demand. Science. 283:496-497
Marcaggi, P. 1999. Capture de NH4+ dans les cellules gliales de rétine d'abeille par un transporteur membranaire spécifique. Thesis No. 698. Université Bordeaux 2.
Marcaggi, P., Coles, J.A. 1998. The major routes of entry of NH4+ and K+ into bee retinal glial cells are independent. J. Physiol. 513:15P-16P. (Abstr.).
Marcaggi, P., Thwaites, D.T., Coles, J.A. 1996. Accumulation of protons in glial cells dissociated from bee retina in response to ammonium. J. Physiol. 495:60P. (Abstr.).
Marcaggi, P., Thwaites, D.T., Deitmer, J.W., Coles, J.A. 1999. Chloride-dependent transport of NH4+ into bee retinal glial cells. Eur. J. Neurosci. 11:167-177[Medline].
Margolis, L.B., Novikova, I.Y., Rozovskaya, I.A., Skulachev, V.P. 1989. K+/H+-antiporter nigericin arrests DNA synthesis in Ehrlich ascites carcinoma cells. Proc. Natl. Acad. Sci. USA. 86:6626-6629[Abstract].
Nagaraja, T.N., Brookes, N. 1998. Intracellular acidification induced by passive and active transport of ammonium ions in astrocytes. Am. J. Physiol. Cell Physiol. 43:C883-C891.
Nett, W., Deitmer, J.W. 1996. Simultaneous measurements of intracellular pH in the leech giant glial cell using 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein and ion-sensitive microelectrodes. Biophys. J. 71:394-402[Abstract].
Overton, E. 1899. Ueber die allgemeinen osmotischen Eigenschaften der Zelle, ihre vermutlichen Ursachen und ihre Bedeutung fur die Physiologie. Vierteljahrsschr. Naturforsch. Ges. Zuerich. 44:88-135.
Pellerin, L., Magistretti, P.J. 1994. Glutamate uptake into astrocytes stimulates aerobic glycolysis: a mechanism coupling neuronal activity to glucose utilization. Proc. Natl Acad. Sci. USA. 91:10625-10629
Pressman, B.C., Harris, E.J., Jagger, W.S., Johnson, J.H. 1967. Antibiotic-mediated transport of alkali ions across lipid barriers. Proc. Natl. Acad. Sci. USA. 58:1949-14956[Medline].
Race, J.E., Makhlouf, F.N., Logue, P.J., Wilson, F.H., Dunham, P.B., Holtzman, E.J. 1999. Molecular cloning and functional characterization of KCC3, a new K-Cl cotransporter. Am. J. Physiol. 277:C1210-C1219
Robinson, R.A., Stokes, R.H. 1959. Electrolyte solutions. 2nd ed London, UK, London Butterworths, pp. 571 pp.
Roos, A., Boron, W.F. 1981. Intracellular pH. Physiol. Rev. 61:296-434
Sanders, D., Hansen, U.-P., Gradmann, D., Slayman, C.L. 1984. Generalized kinetic analysis of ion-driven cotransport systems: a unified interpretation of selective ionic effects on Michaelis parameters. J. Membr. Biol. 77:123-152[Medline].
Sillén, L.G. 1964. Stability constants of metal-ion complexes. I. Inorganic ligands. London, UK, Chemical Society (Spec. Publ. 17.), pp. 150 pp.
Singh, S.K., Binder, H.J., Geibel, J.P., Boron, W.F. 1995. An apical permeability barrier to NH3/NH4+ in isolated, perfused colonic crypts. Proc. Natl. Acad. Sci. USA. 92:11573-11577[Abstract].
Sugden, P., Newsholme, E. 1975. The effect of ammonium, inorganic phosphate and potassium ions on the activity of phosphofructokinases from muscle and nervous tissues of vertebrates and invertebrates. Biochem. J. 150:113-122[Medline].
Szatkowski, M.S., Thomas, R.C. 1989. The intracellular H+ buffering power of snail neurones. J. Physiol. 409:89-101[Abstract].
Thomas, J.A., Buchsbaum, R.N., Zimniak, A., Racker, E. 1979. Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry. 18:2210-2218[Medline].
Thomas, R.C. 1984. Experimental displacement of intracellular pH and the mechanism of its subsequent recovery. J. Physiol. 354:3P-22P[Medline].
Tsacopoulos, M., Poitry-Yamate, C.L., Poitry, S. 1997a. Ammonium and glutamate released by neurons are signals regulating the nutritive function of a glial cell. J. Neurosci. 17:2383-2390
Tsacopoulos, M., Poitry-Yamate, C.L., Poitry, S., Perrottet, P., Veuthey, A.L. 1997b. The nutritive function of glia is regulated by signals released by neurons. Glia. 21:84-91[Medline].
Tsacopoulos, M., Veuthey, A.L., Saravelos, G., Perrottet, P., Tsoupras, G. 1994. Glial cells transform glucose to alanine which fuels the neurons in the honeybee retina. J. Neurosci. 14:1339-1351[Abstract].
Volk, C., Albert, T., Kempski, O.S. 1998. A proton-translocating H+-ATPase is involved in C6 glial pH regulation. Biochim. Biophys. Acta. 1372:28-36[Medline].