Institut für Physiologie, Ruhr-Universität Bochum, D-44780, Bochum, Germany
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ABSTRACT |
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The gastric mucosa is covered by a continuous layer of mucus. Although important for understanding the mechanism of this protective function, only scarce information exists about the pH inside the gastric gland and its outlet. pH in the lumen of the gastric glands, in the outlet of gastric crypts, and in the adjacent cells was measured in the isolated acid-secreting mucosa of the guinea pig. Ultrafine double-barreled pH microelectrodes were advanced at high acceleration rates through the gastric mucus and the tissue to ensure precise intracellular and gland lumen pH measurements. A pH gradient was found to exist along the gastric gland, where the pH is 3.0 at parietal cells, i.e., in the deepest regions, and increases to 4.6 at the crypt outlet. Intracellular pH (pHi) of epithelial cells bordering a crypt outlet, and of neck cells bordering a gland, was acidic, averaging 6.0 and 6.5, respectively. pHi of deep cells bordering a gland was nearly neutral, averaging 7.1, and the secreting parietal cells were characterized by a slightly alkaline pHi of 7.5. This gland pH gradient is in general agreement with a model that we recently proposed for proton transport in the gastric mucus, in which protons secreted by the parietal cells are buffered to and transported with the simultaneously secreted mucus toward the gastric lumen, where they are liberated from the degraded mucus.
crypt pH; epithelial cell pH; ultrafine-tipped pH microelectrodes
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INTRODUCTION |
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THE GASTRIC MUCOSA IS PROTECTED against secreted acid and pepsin by a continuous layer of gastric mucus. We recently demonstrated (10) that the gastric mucus acts both as a diffusion barrier and a proton transport medium. Thus protons secreted by the parietal cells in the gastric glands are buffered by the mucin component of the simultaneously secreted mucus and are transported with this mucus toward the lumen as the mucus layer grows by new mucus formation. Pepsinogen, simultaneously secreted with protons, bicarbonate, and mucus, is converted to pepsin during this transport, partly by spontaneous activation and partly through the action of protons diffusing from the gastric lumen back into the proceeding mucus layer. The pepsin degrades the mucus, and this results in proton release from mucus bonds. The pH profile in the mucus layer represents, therefore, a combination of proton back-diffusion, bicarbonate secretion, and proton release from mucus. Because proton release is irreversible, the protons released are trapped in the gastric lumen, and this explains the asymmetry of the mucus layer as a convective transport medium in the epithelial-to-luminal direction and as a proton barrier in the opposite direction.
This model is in agreement with the stable pH gradient observed across the mucus layer with nearly neutral pH values at the epithelial surface. The model would also predict that the pH values in gastric glands are higher than the luminal pH and that pH should increase from the depth of the gland toward its outlet as mucus and bicarbonate are added to the gland lumen.
Various measurements of pH inside the cells of the gastric mucosa have been reported in isolated gastric glands (9, 11), in the intact frog mucosa (1, 4, 7, 8), and in the rat mucosa in vivo (6). In cells exposed to the cell lumen, the apical membrane was shown to be resistant to acidification (12, 13). However, information on pH in the gastric gland lumen has been reported only very recently. Debellis et al. (3) reported the pH in the frog gastric gland to be as low as 2.5 during stimulation of acid secretion and 7.6 during its inhibition with cimetidine. Chu et al. (2) investigated the pH at the gastric outlets using confocal microscopy and the dye combination of NERF and lucifer yellow in the rat. At a luminal pH of 3 they reported a mucus pH of 4.3 at the epithelial surface and of 5.3 at the gland outlet; at a luminal pH of 5 they reported a pH of 3.9 at the epithelial surface and of 3.5 at the gland outlet. They did not find channels inside the mucus layer and suggested the presence of a mixture of luminal and glandular fluid in the pit, indicated by the appearance of the dye in the pit mucus even though it was initially applied in the luminal fluid.
We report here pH measurements in the lumen of gastric glands and in the cells bordering them. These measurements were made possible by the use of ultrafine double-barreled microelectrodes that were advanced with very high acceleration through the highly viscous mucus, thus preventing dimpling.
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METHODS |
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Guinea Pig Gastric Mucosa Explant
Mucosa preparations from 45 guinea pigs (body mass 350-500 g) were used in these experiments. The stomach was excised and cut into two halves along the lesser and the longer curvatures. The seromuscularis layer was gently removed under continuous drops of ice-cold, oxygen-rich 0.9% NaCl solution. One half of the mucosa was used immediately in the experiment, and the other half was stored in ice-cold, oxygen-rich (95% O2-5% CO2) solution for another type of experiment.Preparation of Gastric Mucosa
The general procedures for measuring pH in the explanted gastric mucus and mucosa were described earlier in detail (10). Briefly, the mucosa corporal/fundal region was fixed in the measuring chamber, which consisted of a closed lower chamber and an open upper chamber with the mucosa separating these parts with an effective tissue area of 2.8 cm2. The mucosa was mounted with the serosal side facing the solution in the lower chamber and was fixed on a net between the chambers by a slightly subatmospheric pressure (The lower (serosal) chamber was continuously perfused at a rate of 10 ml/min with the serosal solution (see below), which was equilibrated with carbogen (95% O2-5% CO2). The upper (luminal) chamber was continuously superfused (5 ml/min) with the luminal solution, which was also equilibrated with carbogen. Both solutions were thermostated at 37°C, and this value was controlled by means of a thermosensor close to the mucosa.
Under these conditions the mucosa secreted acid and mucus for at least 2 h. Even at a luminal pH as low as 1.9, the pH gradient across the mucus layer, to an epithelial pH of 5-6, remained stable for several hours.
Serosal and Luminal Solutions
The serosal solution contained (in mM) 122 NaCl, 5 KCl, 25 NaHCO3, 0.5 MgSO4, 1 KH2PO4, 2 CaCl2, and 20 glucose, with 0.5 g/l Nephrotect AS 10 (mixture of amino acids; Fresenius, Bad Homburg, Germany) and 2.5 vol% FC 43 emulsion (oxygen-carrying perfluorochemical emulsion; Green Cross, Osaka, Japan). The solution was equilibrated at 15°C with carbogen.To stimulate acid secretion, the following substances were added to the serosal solution (in µM): 100 histamine, 0.1 carbachol, and 0.1 pentagastrin. The luminal solution contained 100 mM citrate HCl and 150 mM NaCl adjusted to a pH of 3.0 (30 preparations) or 1.9 (15 preparations).
pH Microelectrodes
For preparation of microelectrodes, double-barreled borosilicate glass capillaries (OD 1.2 mm, ID 0.84 mm, without filament; Hilgenberg, Malsfeld, Germany) were rinsed with distilled water and dried at 300°C for at least 1 day. The capillaries were pulled, using an air jet pipette puller (Sutter Instruments, Novato, CA), to a tip diameter <0.1 µm and a tip angle <5°. For silanization of the pH-reading barrel, the pipettes were vaporized with hexamethyldisilazane (HMDS; Sigma, St. Louis, MO) at 49°C and then heated at 300°C for 60 min. Silanization of the outside of the pipette tip and of the voltage-reading barrel was avoided by fitting the pipette in a holder, which sealed the outside and the voltage-reading barrel against the HMDS vapor.The proton ionophore [Selectophore Hydrogen Ionophore II, Cocktail A: 6 wt% 4-nonadecylpyridine, 93 wt% 2-nitrophenyl octyl ether, 1 wt% potassium tetrakis(4-chlorophenyl)borate; Fluka, Buchs, Switzerland] was introduced under a stereo microscope from the back end into the distal 0.5 mm of the tip of the pH-reading barrel, using a small capillary. The remainder of this barrel was filled with phosphate buffer (pH 7.0) and 150 mM NaCl. The voltage-reading barrel was filled with 150 mM NaCl in black ink. The solution in each barrel was electrically connected by silver/silver chloride wires to a high-impedance voltmeter, which allowed separate recording of either potential as well as of their difference (pH).
All measurements were carried out in a vibration-isolated shielded box. Each microelectrode was calibrated by using standard buffer solutions before the experiment and within the set-up after the measurement. In the range of pH 2-9 the potential difference across the ionophore layer was linearly related to pH, and this relation was in agreement with the Nernst equation.
Microelectrode Holder and Advancement
The microelectrodes were advanced from the luminal side, through the mucus layer, and toward the epithelium, using an acceleration of several 100 g over step distances of <1 µm, with a modified piezo positioner (Märzhäuser, Wetzlar, Germany). For this, the microelectrodes were mounted precisely in the center of a specially designed electrode holder. The piezo positioner and electrode holder were mounted in a vibration-damped micromanipulator of high stability. The high weight (50 kg) of the micromanipulator, with virtually no slackness, the rigid mounting of the piezo crystal, and the firmly locked microelectrode reduced the lateral vibration of the microelectrode tip. This was monitored by a vibration sensor at the piezo crystal during stepping to levels below those that would cause cell destruction. The fitting of the piezo crystal, the electrode holder, and the micromanipulator were constructed in our workshop.pH and Voltage Measurement
Both the pH and the voltage readings of the electrode were continuously monitored; at each microelectrode step the pH signal was allowed to settle and stabilize, which was attained within ~10-20 s. Electrode acceleration was selected to be high while advancing through the mucus but lower in the depth of the tissue, particularly when penetrating cells.The calibration of each microelectrode was verified at the end of the
measurement by recording the luminal pH (1.9 or 3.0; luminal solution)
and the serosal pH (7.4; submucosa). Because of the transepithelial
potential difference (PD) between the luminal and serosal solution,
only one of these solutions could be used as reference. To ensure a
stable offset of the ultrafine-tipped microelectrodes [pH channel
inner resistance (Ri) 1013
], both solutions had to be grounded. Under these
conditions the transmucosal PD between the solutions was reduced. The
advantages were stable pH measurements and an electrical zero potential
inside the gland lumen; the disadvantages were loss of the possibility of measuring transepithelial resistance and potential and uncertainty as to absolute values of membrane potentials. After the bridge between
the solutions was disconnected (bridge resistance 10 M
), the
membrane potential of surface and foveolar cells, related to the lumen,
was about
35 mV and the MP of deep cells, related to the gland lumen,
was about
25 mV.
Because of the slant of its path into the tissue, the microelectrode is expected to cross through the mucosal gastric glands with their bordering epithelial cells and the adjoining interstitium. As indicated, the voltage reading was used to indicate the location of the electrode within the mucus, within the gland lumen, intracellularly within epithelial cells, or (rarely) within the interstitium. During the passage through the mucus, the voltage-reading barrel showed no PD from the reference electrode in the luminal solution, indicating that the entire mucus layer, including mucus within the gastric crypts, is isoelectric with the solution in the gastric lumen. A negative voltage was taken to indicate that the electrode had entered a cell; a stable voltage reading in this situation indicated that the cell remained viable.
Track of Microelectrode in Mucus and Tissue
At the beginning of each track the microelectrode was positioned at the luminal surface of the gastric mucus, which was easily visible under the microscope by the small carbon particles adhering to it. The electrode was advanced at an angle of 30° from the vertical position in steps of 10 µm through the mucus. When the mucus pH (see below) was ~5, this was taken to indicate that the electrode tip approached the epithelial surface. The step size was then reduced to 5 µm and, finally, to 2 µm when the epithelial surface was reached. In Fig. 1, a typical track in the gastric mucosa and in the mucus layer is shown schematically. Five to ten tracks were recorded in a given mucosa.
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The vertical distance of the electrode tip from the luminal surface of the mucus or from the epithelial surface was calculated from the cumulative electrode steps, corrected for the slant. When data are reported on a given electrode track, the actual (slanted) distance is used; but the distance is recalculated for the vertical depth when averaged, calculated data on gland pH are reported.
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RESULTS |
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Recording of pH and Membrane Potential in Several Tracks
Figure 2 shows an example of an electrode track between ~10 µm from the epithelial surface into the mucus and ~50 µm into the mucosal tissue. In this track (as in the recordings of Figs. 3 and 4), the luminal pH was 3.0. Before the recordings in Fig. 2, the electrode had passed through the mucus and approached the epithelial surface at the end of the second minute of recording. The mucus at the epithelial surface is isoelectric with the luminal solution; its pH is ~6.1. At this time and distance, the electrode penetrates a cell, indicated by the negative membrane potential, with an intracellular pH (pHi) value of ~6.7. Advancing the electrode five steps of 2 µm revealed penetration into a second cell of similar properties (e.g., pHi = 6.8). Advancing another 10 µm resulted in an abrupt change of the membrane potential to an isoelectric potential and a drop in pH to somewhat below 5. The recording of both membrane potential and pHi remained stable for ~30 µm of electrode path, before the electrode entered a cell again, with somewhat more negative values of the membrane potential and somewhat less acidic pHi than the initial cells.
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These recordings are interpreted to mean that the electrode had
initially penetrated two cells of similar size (diameter ~10 µm),
membrane potential, and pHi before it entered into the
mucus of the crypt outlet. The electrode, after ~28 µm in the crypt outlet, entered another cell. The inset in Fig. 1 serves to visualize the track of the electrode. The depth of the recording site
(ltiss) relative to the epithelial surface (=
depth in the tissue) can be calculated from the length of the track
from the initial encounter of the epithelial surface to the recording
site (
ltrack), accounting for the slant
(30°) of the track as
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Figure 3 shows a similar recording from regions deeper in the tissue. Again, the electrode recorded pH and membrane potential in two cells before it entered the gland lumen, where the pH in this case was more acidic at ~4. The track length in the gland was only 4 µm, giving a gland width of only 2 µm, much smaller than in the upper, neck region. When the electrode left the gland, the cell penetrated was apparently destroyed by the penetration of the microelectrode, as evidenced by the decline of membrane potential and the slow acidification. However, the next cell was viable, giving potential and pH readings similar to those on the opposite side of the gland.
Figure 4 shows a recording even deeper below the epithelial surface.
Here the electrode recorded initially from a cell that had a somewhat
more negative membrane potential than cells in more superficial regions
and an almost neutral pH. The next cell, however, differed markedly
from all cells so far shown in that it showed a membrane potential of
25 mV and a pH of ~7.5. This cell was interpreted as a parietal
cell, because it is known that acid-producing cells display a more
alkaline pH in their cytoplasm than surrounding epithelial cells
(4). Leaving this cell, the electrode recorded from the
gland lumen, as evidenced again by the zero potential; the pH was even
more acidic than in more superficial recordings. The (corrected) gland
width in this case was also ~2 µm. The next two cells were again
parietal cells.
When the microelectrode was left for several minutes in an area of zero
potential reading and low pH, small oscillations (7-10 min1) with amplitudes of ~1 mV and pH 0.05 were
observed (not shown in Fig. 4). These oscillations were exclusively
observed in this area, and we interpreted them to be myoepithelial
contractions. From these and similar tracks, data can thus be obtained
about the geometry of the gland and its cells and the pH values inside the cells, in the gastric lumen, and in the tissue underlying the epithelium.
Cell Types
On the basis of membrane potential and pHi values, we have classified the cells in which recordings were made into four types. Table 1 compiles the average data obtained for these four cell types.
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Surface epithelial cells. Surface epithelial cells are recorded close to the crypt outlet, i.e., close to the epithelial surface, and are characterized by a cell diameter of ~10 µm and a pHi of 6.0 ± 0.3 (at luminal pH of 3.0). Typically, when one of these cells was destroyed by the penetration of the electrode, the neighboring cell would show a less negative membrane potential and a more acidic pHi, indicating perhaps that neighboring cells are gap junction coupled. Surface epithelial cells were encountered in tissue depths of 0 to 70 ± 25 µm.
Neck cells. The diameter of neck cells is >10 µm; they are characterized by a pHi of 6.5 ± 0.4 (at luminal pH of 3.0) and were encountered in depths of 70 to 150 ± 35 µm. Like surface cells, neck cells were coupled to their neighboring cells, showing the same membrane potential and pH. The borderline between surface and neck cells was detected in each trace as a sharp increase in pH. A single neck cell between surface cells and a single surface cell between neck cells were rare phenomena, and those cells were never separated by more than two intervening cells from their group. We took only one of the surface and neck cells from each trace. For that reason, the majority of recorded cells were excluded from analysis because of their similarity to neighboring cells.
Deep cells. The diameter of deep cells is larger than that of surface and neck cells (~20 µm), and their pHi is less acidic, on average 7.1 ± 0.2 (at luminal pH of 3.0). Deep cells occurred at depths in excess of 150 µm and all the way to the base of the gland (see Parietal cells).
Parietal cells. The diameter of parietal cells (in the acid-secreting mucosa) also is ~20 µm, but the pHi was always alkaline, on average 7.5 ± 0.3. The first parietal cell recorded in a given track was at 210 ± 100 µm. Typically, a significant peak in potential reading was detected when the cell was penetrated. The last cell met on a given track before it entered the interstitium was often a parietal cell, and it occurred at a depth of 275 ± 105 µm; this depth thus constitutes the average length of a gastric gland.
Gland Geometry and pH
From the single tracks it was apparent that both gland pH and diameter declined with depth into the gastric gland. We have used a normalized gastric gland to plot the individual measurements along the gland. For this, we have corrected the length of any given gland to the mean gland length of 275 µm (see Parietal cells); any given recording site was corrected with the same correction factor as the gland length.Figure 5 shows the individual data
points. There is a rather steep slope of the pH profile into the gland,
from ~4.6 at the crypt outlet to ~3.0 in the parietal cell region.
The gland diameter again shows a substantial scatter. However, it is
apparent that the gland width narrows quickly with depth from the
outlet and then reduces further at a much lower pace. Although some
wider diameters have been recorded at the final depth, most glands show a diameter of 1-4 µm.
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Dependence on Luminal pH
The pH profiles over most parts of the gastric mucus were different between the two luminal pH values tested, 3.0 and 1.9, as we reported earlier (10). However, in layers closer than 50 µm from the epithelial surface the profiles became nearly identical, and, indeed, the pHi values of all four cell types did not differ significantly between the two luminal pH values (see Table 1), even though a tendency was obtained for slightly lower pHi values at the lower luminal pH.Summary of pH Values in Gastric Gland
The average data for mucus pH, above the epithelium and in the gastric gland, and the pHi of adjacent cells, when luminal pH is 3.0, are plotted in Fig. 6. From the acidic gastric lumen, the mucus becomes more alkaline as the epithelial surface is approached; moving into the gland, the gland mucus becomes more acidic with depth. However, nowhere is the mucus pH significantly below 3, and this is true even when the luminal pH is below this pH value.
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DISCUSSION |
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Critique of Methods
Microelectrode measurement. pH measurements inside epithelial cells in the secreting intact mammalian mucosa are difficult to perform. We had to penetrate several hundred micrometers of the highly viscous gastric mucus and, depending on the depth, an additional cell layer before entering a cell. In addition, there remained the danger of damaging the cell with the microelectrode. We have reduced this difficulty by using a vibration-free setup and a serosal solution flow without any movement of the tissue and by ensuring a high acceleration of the advancing microelectrode (see Dimpling of the mucus layer). We have only analyzed cells with a stable membrane potential and a stable pH recording.
The use of intracellular dyes in the analysis can be limited by the possibility of visualizing exfoliated epithelial cells in the gastric mucus; these cells occur in several layers but cannot be regarded as active and physiologically intact. We noticed in nearly every trace that the tip of the microelectrode was not visible through the microscope below these layers of exfoliated cells before it reached the active epithelial layer, where cells were encountered with a stable membrane potential and pHi in the physiological range. Therefore, the advantage of direct microelectrode measurement appears to be the ability to discern between living and exfoliated cells in the intact gastric mucosa.Dimpling of the mucus layer. The mechanical resistance of the mucus layer was much higher than the resistance of the gastric tissue. With conventional microelectrodes, the step size of the electrode would be larger than the distance that was penetrated by the microelectrode tip during the step (dimpling). We have used ultrafine-tipped microelectrodes and a high level of acceleration to circumvent these problems. To estimate the remaining dimpling, the mucus/lumen surface was marked by small carbon particles. In an ideal case, the position of the carbon particles should move only slightly during penetration. We have analyzed only those tracks in which the particle movement was small and have attempted to partially correct our depth reading using the carbon particle movement.
Identification of gastric glands. Depending on the depth of the microelectrode tip, the gastric glands were only a few micrometers wide (see Fig. 4). In deeper gland regions, the gland lumen is thus extremely small compared with the size of the bordering cells and their lamina propria. This had two consequences for measuring inside a gastric gland. One consequence is that the electrode sometimes passed through the gland in a single step, leaving one cell and immediately entering another one on the opposite side. The second consequence is that the probability of meeting a gland during a given track was not very high, and, indeed, many tracks were not successful in this respect.
Identification of parietal cells.
Several types of evidence led us to identify parietal cells; for
example, they are located deep in the gastric gland and they have a
relatively large cell diameter. Furthermore, in several instances we
have been able to access a parietal cell from the gland lumen, thus
showing their bordering property and the acidic gland pH in their
vicinity. Also, the high pHi values, compared with those of
the other gland cells, were taken to indicate identity as parietal
cells. In the frog mucosa (4), cells with an intracellular alkalization on histamine stimulation have been taken to be parietal cells by virtue of a HCO3/Cl
exchanger.
Whether parietal cells in our preparation with normal blood supply
would exhibit an alkaline pHi remains, however, unknown.
Gland pH Gradient and Proton Transport
Our finding of a pH profile along the gastric gland is in general agreement with the model that we proposed previously (10) for proton transport by the gastric mucus. In this model, the protons secreted by the parietal cells would be reversibly bound by the mucus proteins and would be transported with the mucus toward the gastric lumen. On the basis of this model, recognizing the occurrence of parietal cells largely in the deepest regions of the gland, one would expect the pH to be lowest in gland lumen at this site. As more mucus and bicarbonate are produced in higher crypt regions, the buffering potential would increase and the pH thus increase as well. At the crypt outlet the pH would then be least acidic, thus protecting the epithelial surface of the mucosa.Thus the pH profile clearly reveals the fact that protons are buffered within the gastric gland. If protons were transported in channels through the mucus layer (5), the pH inside the gastric glands would have to be lower than the luminal pH and this low pH would have to be present in the crypt outlet as well as in the parietal cell region.
Gland pH and Luminal pH
The pH profile in the gland as well as the pHi of the adjacent cells were very similar at luminal pH values of 3.0 and 1.9. Indeed, the differences were largely compensated by different pH gradients in the mucus layers, thus rendering the pH at the epithelial surface nearly identical in both situations. Although this shows the effective protective function of the gastric mucosa in controlling the epithelial pH, it should be appreciated that we have used the same maximal stimulation of acid secretion in both situations. Under physiological conditions, these profiles may be somewhat different because the mucosa is expected to secrete fewer protons at the more acidic pH.Our pH results at the epithelial surface and in the gland outlet are similar to those reported by Chu et al. (2). However, there are at least two details in which their report differs from ours. One difference is that these authors found the gland outlet pH to vary with luminal pH, and the other is that they found the pH gradient across the mucus to be small or even absent. We do not have an explanation for this apparent discrepancy; it requires further experimentation.
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ACKNOWLEDGEMENTS |
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This work was supported by the Deutsche Forschungsgemeinschaft (Grants Sche 46/14-1 and 46/14-2) and by the Medizinische Fakultät, Ruhr-Universität Bochum.
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FOOTNOTES |
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Address for reprint requests and other correspondence: S. Schreiber, Institut für Physiologie, MA 2/149, Ruhr-Universität Bochum, D-44780, Bochum, Germany (E-mail: Soeren.Schreiber{at}ruhr-uni-bochum.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 26 August 1999; accepted in final form 2 February 2000.
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