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Departments of 1 Physiology and 2 Medicine, Dartmouth Medical School, Lebanon, New Hampshire 03756
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We used 2',7'-bis(carboxyethyl)-5(6)-carboxyflourescein (BCECF), a pH-sensitive fluorescent dye, to study intracellular pH (pHi) regulation in neurons in CO2 chemoreceptor and nonchemoreceptor regions in the pulmonate, terrestrial snail, Helix aspersa. We studied pHi during hypercapnic acidosis, after ammonia prepulse, and during isohydric hypercapnia. In all treatment conditions, pHi fell to similar levels in chemoreceptor and nonchemoreceptor regions. However, pHi recovery was consistently slower in chemoreceptor regions compared with nonchemoreceptor regions, and pHi recovery was slower in all regions when extracellular pH (pHe) was also reduced. We also studied the effect of amiloride and DIDS on pHi regulation during isohydric hypercapnia. An amiloride-sensitive mechanism was the dominant pHi regulatory process during acidosis. We conclude that pHe modulates and slows pHi regulation in chemoreceptor regions to a greater extent than in nonchemoreceptor regions by inhibiting an amiloride-sensitive Na+/H+ exchanger. Although the phylogenetic distance between vertebrates and invertebrates is large, similar results have been reported in CO2-sensitive regions within the rat brain stem.
respiratory control; acid-base balance; central carbon dioxide chemoreceptors; invertebrates; snails
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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SINCE WINTERSTEIN FIRST PROPOSED his "reaction theory" of respiratory control in 1910 (32, 33) in which he attributed the excitatory effects of CO2 on ventilation to changes in hydrogen ion concentration, investigators have debated both the validity of the theory and the locus of excitation. Two issues pertain to the locus of excitation: where are CO2 chemoreceptors within the central nervous system and where is the pH that the chemosensors detect [extracellular pH (pHe), intracellular pH (pHi), the pHe-pHi gradient, etc.]? We have explored these issues in an air-breathing invertebrate, Helix aspersa (12). Snails are phylogenetically distant from mammals, and aerial respiration evolved independently in vertebrates and invertebrates. Nonetheless, pulmonate, terrestrial snails developed remarkably similar central neural mechanisms to monitor CO2 and regulate ventilation as a function of CO2 and pHi (9-11, 13, 16). For example, exposure of the whole snail to CO2 increased opening of the pneumostome, a muscular aperture that regulates access to the gas exchange surface of the mantle cavity. Furthermore, we identified a discrete, CO2-sensitive region along the margins of the visceral and right parietal ganglia in the central nervous system of the snail that mediated responses of the pneumostome to CO2. Focal hypercapnic stimulation of this CO2-sensitive region increased pneumostomal opening and mimicked the response that we observed in intact snails exposed to ambient hypercapnic gases (11). We also identified intrinsically CO2-sensitive neurons within the CO2-sensitive region (14).
Within identified CO2 chemoreceptor regions, the location of the "CO2 receptors" (intracellular vs. extracellular) has not been defined in either vertebrates or invertebrates (17, 19). In H. aspersa, pHi, as opposed to pHe or the pHe-pHi gradient, seems to be the essential stimulus of CO2 chemoreceptors (10). The evidence is less clear-cut in mammals, but available data are consistent with the hypothesis that mammalian CO2 chemoreceptors also respond to pHi (19). If pHi mediates the ventilatory effects of CO2, pHi regulation may differ between chemoreceptor and nonchemoreceptor cells. In theory, chemoreceptor neurons, unlike other cells, should not exhibit pHi recovery on acidification with CO2 so that the CO2-induced pHi change, the respiratory stimulus, does not diminish over time; the chemoreceptor stimulus should persist as long as the acidosis persists. On the other hand, robust pHi regulatory mechanisms may exist in nonchemoreceptor neurons to restore pHi during acidic stress and preserve protein and cellular function. Ritucci et al. (24) recently tested this hypothesis when they investigated the effects of hypercapnia on pHi regulation of neurons in medullary brain slices from preweanling Sprague-Dawley rats. Regulation of pHi differed between neurons in chemosensitive areas, the nucleus of the solitary tract (NTS) and ventrolateral medulla (VLM), and nonchemosensitive areas, the inferior olive and hypoglossal nucleus of the medulla. A subset of neurons in the chemosensitive areas was unable to regulate pHi when pHe and pHi fell during acidic stimulation; whereas pHi in neurons in nonchemosensitive areas recovered toward the initial, control pHi although the acidic stress persisted. However, pHi recovered in all areas during intracellular acidosis if pHe was not acidified. Furthermore, pHi recovery from acidic stress in medullary neurons, whether in chemosensitive or nonchemosensitive regions, was due solely to an Na+/H+ exchange mechanism. These results support the hypothesis that chemoreceptor cells have relatively poor pHi regulation but also indicate that the pattern of pHi regulation was highly dependent on pHe. These findings are similar to the pattern of pHi regulation during hypercapnia in isolated glomus cells of the carotid body from neonatal rats, which are also CO2 sensitive (5, 6).
In this study, we compared pHi regulatory function between neurons in the CO2 chemosensitive region and neurons in nonchemosensitive regions in the subesophageal ganglia of H. aspersa. We measured the pHi of individual neurons using the pH-sensitive dye 2',7'-bis(carboxyethyl)-5(6)-carboxyflourescein (BCECF). Individual cellular responses to three different methods of inducing intracellular acidosis were studied: 1) pHe and pHi were varied by hypercapnic acidification, 2) pHe was held constant, whereas intracellular acidosis was induced using the ammonia prepulse method, and 3) pHe was held constant, whereas intracellular acidosis was induced using isohydric hypercapnia. We also examined the pHi regulatory mechanisms whereby neurons within the subesophageal ganglia responded to intracellular acidosis.
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MATERIAL AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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H. aspersa were purchased throughout the year (Pennsylvania Snail) and maintained in a humidified aquarium at 22°C. The snails were fed carrots, lettuce, cucumbers, and cornmeal as previously described (11).
Solutions. Control saline consisted of (in mM) 85 NaCl, 4 KCl, 7 CaCl2, 5 MgCl2, buffered with 20 HEPES (HEPES free-acid; Sigma, St. Louis, MO) and titrated with NaOH to pH 7.8. The hypercapnic solutions contained (in mM) 20 NaHCO3, 90 NaCl, 4 KCl, 7 CaCl2, 5 MgCl2, 0.2 NaH2PO4 equilibrated with CO2 to pH 7.5 (5% CO2) or 7.2 (10% CO2). The ammonia prepulse solution contained (in mM) 10 NH4Cl, 75 NaCl, 7 CaCl2, 5 MgCl2, 4 KCl, 0.2 NaH2PO4 buffered with 20 HEPES free-acid and titrated with NaOH to pH 7.8. Without HEPES in solution, we had persistent difficulties preventing CaCO3 precipitation at room temperature even with added 0.2 NaH2PO4, just as Thomas described (27). In addition, we did not use control solutions for NH4Cl perfusion in which Na+ was held constant by substituting N-methyl-D-glucamine. N-methyl-D-glucamine-containing solutions were also not stable at pH 7.8 and room temperature. The isohydric hypercapnic solution consisted of (in mM) 40 NaHCO3, 60 NaCl, 7 CaCl2, 5 MgCl2, 4 KCl, 0.2 NaH2PO4 buffered with 20 HEPES free-acid and equilibrated with CO2 to pH 7.8 (5% CO2). In isohydric solutions containing inhibitors, amiloride (1 mM; Sigma) and DIDS (20 µM; Sigma) were used. The sodium-free BCECF calibration solution consisted of (in mM) 110 KCl, 7 CaCl2, 5 MgCl2 buffered with 10 HEPES free-acid and titrated with KOH to pH 7.2. The acetoxy-methyl ester of BCECF (Molecular Probes, Junction City, OR) was prepared as a 3.4-mM stock solution in DMSO (1 mg/500 µl) and diluted to 30 µM (35.2 µl/4 ml) in control saline. Nigericin (Molecular Probes) was prepared as a 27.5-mM stock solution in DMSO (10 mg/500 µl) and diluted to 16 µM (59.6 µl/100 ml) in the calibration solution. The osmolality of all the solutions was 225 ± 5 mosmol/kgH2O.
Isolated central nervous system preparation. The subesophageal ganglia and the cerebral ganglia were removed after sectioning all neural connectives and the aorta as described previously (11). The isolated central nervous system was pinned with the dorsal surface exposed in a perfusion chamber contained within a petri dish. The subesophageal ganglia were covered by a thick outer sheath and a thin inner sheath lying directly on and within the neurons of the ganglia. The outer sheath was removed manually and the inner sheath was treated with protease (1 mg/ml; Sigma) for 8 min and delicately pulled away. The protease was rinsed from the chamber by repeated washings with control saline. The isolated central nervous system was incubated in control saline with 30 µM BCECF at room temperature (22°C) for 1.25 h in the dark. A coverslip was placed over the perfusion channel to create a uniform plane of vision and to ensure even perfusion over the isolated central nervous system. The isolated central nervous system was washed with control saline for 10-15 min to remove any remaining extracellular BCECF. Test solutions perfused the bath via gravity-fed tubing at a rate of 10 ml/min. The perfusion chamber was relatively large, and complete solution changes required ~30 s. A small pH electrode ("Beetrode," World Precision Instruments, Sarasota, FL) was used to confirm that the effluent pH from the perfusion chamber was equivalent to the pH entering the chamber.
Imaging of BCECF-loaded neurons. After preparation, the dish was placed under an Optiphot-2 upright microscope (Nikon, Melville, NY) mounted with a SenSys charge-coupled device (CCD) camera (Photometrics, Tucson, AZ) connected to a Dimension XPS computer (Dell Computer, Austin, TX). Neurons on the subesophageal ganglia were excited for ~300-500 ms with light from a 75-W xenon arc lamp (Interlight, Hammond, IA) that was filtered (440 and 500 nm) using a Lambda 10-2 filter wheel (Sutter Instrument, Novato, CA). Emitted light was captured by the CCD camera after passing through a dichroic mirror with a high pass cutoff of 515 nm and a 530 ± 12.5-nm emission filter (Chroma Technology, Brattleboro, VT). We used Axon Imaging Workbench (Axon Instruments, Foster City, CA) to control the filter wheel and collect and process the data.
Calibration of pHi from BCECF fluorescence.
pHi was measured from the ratio of BCECF-emitted
fluorescence after excitation at 500 and 440 nm. A calibration curve of
pHi as a function of normalized fluorescence ratios
(Nfl; normalized to pH 7.2) was calculated as described by
Boyarsky et al. (4). Neurons were perfused with solutions
of known pHe ranging from 6.5 to 8.5, and pHi
values were measured after equilibration between pHe and
pHi using the high K+/nigericin technique. From
calibration of pHi as a function of Nfl, a
calibration curve to transform Nfl ratios into
pHi was constructed using the following equation:
pHi = 7.2073 + log [(Nfl 
0.55378)/(1.45378 Nfl)];
r2 = 0.98; n = 67. A
single-point calibration (pH 7.2, Nfl = 1.0) was
performed at the conclusion of each experiment, and pHi
values were determined from the calibration curve.
pH response protocols.
All experiments were conducted at room temperature (~22°C). Only
neurons in which BCECF fluorescence at 440 nm diminished <0.5%/min
over the course of an experiment were analyzed. BCECF is a vital dye,
and a low leakage rate is an indicator of cell viability. Ritucci et
al. (23, 24) pointed out that pHe
seemed to control the effectiveness of pHi regulation.
Therefore, we designed protocols to reduce pHi while
pHe was reduced or held constant. During hypercapnic
acidosis, CO2 readily penetrates the intracellular space
and pHe and pHi both fall. Two levels of
hypercapnic acidosis were studied to establish a dose-response relationship, pHe 7.5, 5% CO2, and
pHe 7.2, 10% CO2. During ammonia prepulse,
pHe is held constant throughout the protocol, although pHi falls after NH4Cl is removed from the
perfusate (3). We selected a concentration of
NH4Cl and an NH4Cl perfusion time that
generated an intracellular acidosis equivalent to the fall in
pHi associated with milder hypercapnic acidosis
(pHe 7.5, CO2 5%). In the isohydric
hypercapnic experiment, pHe was constant and
pHi dropped. The extracellular HCO3
concentration was raised to keep pHe constant when the
CO2 was raised to 5%. However, CO2 penetrated
the cell and created an intracellular acidosis. In a final set of
studies, the effects on pHi regulation of amiloride (1 mM),
DIDS (20 µM), and combined amiloride (1 mM) and DIDS (20 µM) were
investigated after a rate of pHi recovery had been
established during perfusion with inhibitor-free isohydric hypercapnia.
Analysis and statistics.
We wanted to compare the pattern of pHi regulation of
individual neurons in the chemosensitive and nonchemosensitive areas during acidic stimulation. We measured pHi in neurons from
all ganglia on the dorsal surface of the subesophageal ganglia: the right and left parietal ganglia and the visceral ganglion. In each
experiment, we chose the cells that had the best BCECF filling without
regard to the location of the neurons. We defined the chemoreceptor
region as the upper visceral, right visceral, and left side of the
right parietal ganglia (see Fig. 1), an
area that slightly exceeded the size of the chemoreceptor region we found previously (10, 14). Nonchemoreceptor
cells were defined as neurons in all other regions. When in doubt about
the exact location of a neuron with respect to the chemoreceptor area,
we defined the neuron as chemosensitive. This less-stringent definition of the chemoreceptor region was selected to provide a more stringent test of the null hypothesis, no difference in pHi
regulatory patterns between chemoreceptor and nonchemoreceptor regions,
because we were more likely to include nonchemosensitive neurons within
the chemosensitive area.
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0.05) and positive slope of the regression
of pHi on time. We did not use the presence of
pHi overshoot after removal of the acidic stress as a
criterion of pHi recovery. Measurements of pHi
within each neuron were sufficiently variable that in ~10% of cells
studied in which there was a significant positive slope of
pHi recovery, we could not tell whether overshoot was
present or not, the difference between the control and postacidic
pHi was too small to discriminate with confidence. Hence,
we usually saw overshoot but relied on a more quantitative analysis of
the pHi recovery slope to define significant or
nonsignificant pHi recovery. Unless otherwise stated, all
values are means ± SD.
Each experiment consisted of two acidic stimuli (2 levels of
hypercapnic acidosis, hypercapnic acidosis and ammonia prepulse, or
hypercapnic acidosis and isohydric hypercapnia) and two levels of
pHi were compared within each treatment: the initial,
lowest pHi measured within 3 min of exposure to each
treatment and a recovery pHi normalized to a constant
duration of recovery (1 h). The actual recovery period was variable
among neurons and usually lasted 15-25 min. We used a two-way
ANOVA in which the region (chemoreceptor vs. nonchemoreceptor) was a
between-subjects factor and type of acidic stimuli and pHi
level (initial vs. recovery) were within-subjects factors. In the
analysis of drug effects on pHi during isohydric
hypercapnia, a similar ANOVA was used, but there were two
between-subjects factors: region and drug treatment (amiloride, DIDS or
combined amiloride, and DIDS). The within-subjects factors, type of
treatment (isohydric hypercapnia with or without drug) and
pHi level (initial vs. recovery), remained the same. When
the results of an ANOVA indicated that significant differences existed
among treatment conditions, specific preplanned comparisons were made
after adjusting P values by the Bonferroni method to keep
the overall P value in each experiment at P 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Hypercapnic acidosis.
An example of the protocol and the pHi response of a single
neuron from the chemoreceptor region on the dorsal surface of the
subesophageal ganglia is shown in Fig. 2.
Each experiment began with measurements of pHi during
perfusion with control saline at pH 7.8 and no added CO2. A
pHe of 7.8 is within the normal range of hemolymph pH in
intact, active snails (7). Two levels of hypercapnic
acidosis were studied: pHe 7.2, 10% CO2 and
pHe 7.5, 5% CO2 (the normal hemolymph
CO2 concentration is ~2.5%; Ref. 7). The order
of testing pHe 7.2 and pH 7.5 was varied, but in the
example shown in Fig. 2, the pHe 7.5, 5% CO2
was studied first. The pHi fell quickly after each
hypercapnic stimulation began, and pHi fell more when
pHe was 7.2 compared with pHe 7.5. The rate of
pHi recovery was not significantly different from zero at
either level of hypercapnic acidosis.
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0.113 ± 0.061 pH units/h, respectively.
pHi recovery rates were faster in the nonchemoreceptor
regions at both pHe values: at pHe 7.5 and 7.2, pHi recovery rates were 0.279 ± 0.069 and 0.053 ± 0.063 pH units/h, respectively. Hence, pHi recovery was slower in the chemoreceptor region compared with the nonchemoreceptors at both pHe 7.5 and 7.2 (P < 0.01), and
pHi recovery was slower in both chemoreceptor and
nonchemoreceptor regions at pHe 7.2 compared with
pHe 7.5 (P < 0.01). In the chemosensitive
area, 6 of 20 neurons tested recovered at 5% CO2 and 2 of
20 neurons recovered at 10% CO2. In the nonchemosensitive
area, 12 of 19 neurons tested recovered at 5% CO2 and 10 of 19 neurons recovered at 10% CO2. Hence, pHi
recovery during hypercapnic acidosis was significantly more frequent
among neurons from nonchemoreceptor areas compared with the
chemoreceptor region (
2 = 4.31;
P < 0.04 analyzing only the 5% treatment level).
However, the patterns of pHi recovery were not perfectly
segregated between chemoreceptor and nonchemoreceptor regions: small
numbers of neurons within the chemoreceptor area demonstrated
pHi recovery, and a larger number of neurons in
nonchemoreceptor regions failed to manifest significant pHi
recovery.
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Ammonium chloride prepulse protocol.
After ammonia prepulse, pHi regulatory mechanisms were
studied in chemoreceptor and nonchemoreceptor regions, whereas
pHi changed, but pHe was held constant. An
example of the progression of pHi during an ammonia
prepulse experiment from a single neuron in the nonchemoreceptor part
of the right parietal ganglion is shown in Fig.
4. Each experiment began with a control
measurement of pHi at pHe 7.8. The pattern of
pHi regulation during hypercapnic acidosis (pHe
7.5, 5% CO2) was determined, and this was followed by
NH4Cl exposure (10 mM) at pHe equal to 7.8 for
10 min. After NH4Cl was removed from the perfusate,
pHe was kept at 7.8. In the cell shown in Fig. 4,
pHi was 7.4 when pHe was 7.8 during the
control period. This neuron demonstrated significant pHi
recovery during hypercapnic acidosis (recovery rate equal 0.703 pH
units/h; P < 0.001). When returned to
pHe 7.8 and no CO2, there was an alkaline
overshoot, which was a further manifestation of pHi
recovery during hypercapnic acidosis. During NH4Cl
perfusion, the cell was alkalinized, but pHi fell once
NH4Cl was removed from the perfusate and pHi
started to return toward the control pHi value almost
immediately.
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0.045 ± 0.088 pH units/h, in the chemoreceptor region during hypercapnic acidosis. The recovery rate increased to
0.550 ± 0.159 pH units/h in the same neurons during ammonia prepulse acidification at pHe equal to 7.8. A similar
change occurred in the nonchemoreceptor regions: pHi
recovery was 0.262 ± 0.053 pH units/h during hypercapnic acidosis
and increased to 0.737 ± 0.096 pH units/h during the ammonia
prepulse acidification phase. The pattern of pHi recovery
rate was similar in chemoreceptor neurons and nonchemoreceptor neurons;
the slope of the pHi recovery was less in chemoreceptor
region neurons during both treatment conditions (hypercapnic acidosis
and after NH4Cl), but the difference in slopes between
chemoreceptor and nonchemoreceptor regions failed to reach statistical
significance in the ANOVA (P = 0.056). However, the
pHi recovery rate was significantly greater during ammonia prepulse acidification compared with hypercapnic acidosis in both chemoreceptor and nonchemoreceptor regions (P < 0.001). Finally, the presence or absence of NaHCO3 in the
perfusate did not alter the rate of pHi recovery after the
ammonia prepulse (data not shown).
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Isohydric hypercapnia.
Isohydric hypercapnia is an alternative mechanism to the ammonia
prepulse protocol, whereby pHe remains constant while
pHi is reduced. The increased CO2 present
during hypercapnia quickly diffuses into the neuron and acidifies the
intracellular space, but the pHe is held constant because
the increase in CO2 in the extracellular fluid is matched
by increased bicarbonate. This method has the further advantage that
bicarbonate and CO2 are present during the entire protocol.
An example of this protocol and the response of a single chemoreceptor
neuron are shown in Fig. 6.
pHi in the neuron shown in Fig. 6 dropped from a control pHi value of 7.4 when pHe was 7.8 to a
pHi of ~7.13 when pHe was 7.5. There was no
evidence of recovery of pHi during hypercapnic acidosis.
During isohydric hypercapnia, pHi did not fall quite as low
(pHi ~7.17) as it had when exposed to equivalent
hypercapnia during the hypercapnic acidosis exposure, but
pHi recovered steadily during isohydric hypercapnia.
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Pharmacological studies of pHi regulatory mechanisms.
Amiloride inhibits Na+/H+ exchange
(2), and DIDS is a chloride channel inhibitor that blocks
Cl-dependent HCO3 exchange
(8). We studied the effect of both drugs on the rate of
pHi recovery in neurons in the chemoreceptor region and
nonchemoreceptor regions. After stabilization of pHi in
control saline (pHe 7.8), the neurons were exposed to
hypercapnic and acidic saline (pHe 7.5, 5%
CO2) to determine the pattern of pHi regulation
when both pHe and pHi were changed.
Subsequently, each neuron was also exposed to isohydric hypercapnia
(pHe 7.8, 5% CO2) or isohydric hypercapnia with amiloride (1 mM) or DIDS (20 µM) or both amiloride (1 mM) and
DIDS (20 µM). This concentration of DIDS was selected because it
modified pneumostomal activity in previous studies (10)
and comparable concentrations of SITS inhibited
Na+-dependent Cl/HCO3
exchange in H. aspersa (29, 30).
The order of these treatments (isohydric hypercapnia with or without
drug) was varied. We studied the drug effects during isohydric
hypercapnia to increase the number of neurons with significant rates of
pHi recovery, and we analyzed only neurons that
demonstrated a significant rate of pHi recovery in the
absence of drug treatment. We made this selection to avoid difficulties
determining whether amiloride and DIDS altered the rate of
pHi recovery in neurons with extremely slow rates of
recovery. Of 34 neurons studied in the chemoreceptor region, 8 were
excluded, and of 19 neurons from nonchemoreceptor regions, 3 were
excluded on the basis of slow rates of pHi recovery. The
rates of recovery were less in chemoreceptor region neurons compared
with neurons from nonchemoreceptor regions, but the pattern of
responses to amiloride and DIDS was not significantly different between
regions. Therefore, the data from all regions were combined, and the
pattern of pHi recovery during hypercapnic acidosis was dropped from the analysis of drug effects. The average responses of the
neurons analyzed are shown in Fig. 8. The
initial pHi values during isohydric hypercapnia with and
without drug treatment were not different among treatment groups.
Furthermore, the rates of pHi recovery in the absence of
the particular drug treatment were not different among drug treatment
groups. The rate of pHi recovery during exposure to DIDS
(0.358 ± 0.159 pH units/h) was not different from the
pHi recovery rate during the control isohydric hypercapnia exposure without DIDS (0.325 ± 0.125 pH units/h). Amiloride,
however, caused a significant decrease in pHi recovery rate
(0.355 ± 0.342 pH units/h) compared with the control rate
(0.336 ± 0.177 pH units/h; P < 0.05) in the same
neurons and compared with the DIDS-treated neurons (P < 0.05). Amiloride plus DIDS caused a further significant drop in
pHi recovery rates (0.757 ± 0.418 pH units/h)
compared with the control recovery rate in the same neurons (0.269 ± 0.212 pH units/h; P < 0.05). The pHi
recovery rate during amiloride plus DIDS was also significantly less
than the recovery rate with amiloride alone (P < 0.05). We repeated this analysis on the neurons in the chemoreceptor
region alone, and the results were identical: no effect of DIDS alone,
reduced recovery rates after treatment with amiloride, and a greater
reduction in pHi recovery rates after treatment with
amiloride and DIDS.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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We compared pHi regulation in neurons within the
CO2 chemoreceptor region to pHi regulation in
nonchemoreceptor regions on the dorsal surface of the
subesophageal ganglia of H. aspersa. We biased the
experiment toward finding no differences between areas by broadly
defining the chemoreceptor region as the upper and right quadrant of
the visceral ganglion and left quadrant of the right parietal ganglion.
Nonchemoreceptor cells were defined as neurons in all other regions on
the dorsal surface of the subesophageal ganglia. Despite this generous
definition of the chemoreceptor regions, neurons in the chemoreceptor
region were, on average, less able to regulate pHi under
all acidic stimuli tested, although the initial pHi in all
conditions tested was similar among chemoreceptor and nonchemoreceptor
areas. Furthermore, pHi regulation was less effective in
all neurons when the acidic stimulus was associated with a drop in
pHe. The dominant pHi regulatory mechanism is
probably an amiloride-sensitive Na+/H+
exchanger, but there may be a small role for a DIDS-sensitive Cl/HCO3 exchange mechanism. These
results are, in general, strikingly similar to results from neurons in
CO2-sensitive regions in the brain stem of preweanling rats
(23, 24) despite the independent evolution of
aerial respiration in vertebrates and invertebrates.
Control and initial pHi values. The pHi of chemosensitive and nonchemosensitive neurons was not significantly different under steady-state conditions (control saline, pH 7.8) in any of our experiments. The resting steady state pHi varied between 7.4 and 7.5. This value is similar to pHi values (7.41 ± 0.08; mean ± SD) described previously by Thomas (28) using intracellular pH electrodes in neurons in H. aspersa. We studied three acidic stimuli: hypercapnic acidosis, ammonia prepulse, and isohydric acidosis, and in all cases, the initial pHi values measured within 3 min of applying each stimulus were similar among neurons from the chemoreceptor region and nonchemoreceptor regions. Therefore, any differences in pHi recovery (see below) cannot be attributed to differences in the initial intracellular or extracellular pH. However, the lack of differences in pHi among chemoreceptor and nonchemoreceptor regions during acidic stimulation is at odds with some previous work. When pHe was changed from 7.48 to 7.30 in the experiments described by Ritucci et al. (24), pHi fell by ~83% of the fall in pHe in the NTS; the reduction in pHi was ~33% of the fall in pHe in the VLM. In contrast, pHi fell by only 4-22% of the change in pHe in nonchemosensitive regions (the inferior olive and hypoglossal nucleus). In isolated glomus cells of the rabbit carotid body, which are CO2 sensitive, pHi fell by ~60-70% of the change in pHe during hypercapnic acidosis (5). The usual change in pHi is ~20-30% of the change in pHe in other nonchemosensitive tissues (see Ref. 5 for a complete list of references). In the snail neurons, the change in pHi was 71% of the change in pHe when pHe changed from 7.5 to 7.2 during hypercapnic acidosis, comparable to the results in the NTS and in carotid body glomus cells. However, the change in pHi for this change in pHe was not different between chemoreceptor and nonchemoreceptor cells.
The rate of pHi recovery was slow (see below), and we estimated the buffering capacity in these neurons without including drugs to inhibit proton or bicarbonate exchange. We calculated the changes in intracellular HCO3 associated with the
measured changes in pHi apparent within 3 min of changing
from control saline (pH 7.8, nominally CO2 free) to either
pH 7.5 (5% CO2, 20 mM NaHCO3) or pH 7.2 (10%
CO2, 20 mM NaHCO3) using an apparent pKa of
carbonic acid and a CO2 solubility coefficient derived from
pulmonate snail hemolymph (1). The estimated buffering
capacity at pH 7.5 was 17.5 ± 11.5 and 50.7 ± 25.7 meq
H+/pH unit at pH 7.2. These values are similar to those
described by Thomas (27) in nonchemosensitive neurons
using the same method. The particular value of the buffering capacity
is of less interest in our study, however, than the lack of any
difference in buffering capacity between chemoreceptor and
nonchemoreceptor regions. Buckler et al. (5) indicated
that a steep pHi vs. pHe relationship was
present in cells that acted as sensitive pH detectors. However, we
found no such relationship in the neurons we studied: neurons in
nonchemoreceptor areas had pHi vs. pHe
relationships as steep as neurons in the chemoreceptor region. The
nonchemoreceptor regions might have some nonrespiratory chemoreceptor
function, but the ubiquity of the steep pHi vs.
pHe relationship in the neurons that we studied leads us to
conclude that a steep pHi vs. pHe relationship
is a necessary, but not sufficient, marker of pH sensitivity.
Intracellular pH regulation. Three main points emerge from the studies of the rate of pHi regulation. First, regulation of pHi during acidic stress was slower and less effective in neurons from the chemoreceptor region in all conditions studied. Second, the rate of pHi regulation was slower in all regions when pHe was reduced compared with acidic stresses of equal intracellular severity but constant pHe. Finally, the inhibitory effect of pHe on the rate of pHi recovery was graded: the lower the pHe, the slower the rate of pHi recovery. The actual rates of pHi recovery that we observed were similar to those reported by Thomas (30) in H. aspersa, but slightly slower than the recovery rates reported by Ritucci et al. (24) in rat neurons studied at 37°C. During hypercapnic acidosis at both pHe 7.5 and 7.2, pHi regulation was significantly slower in neurons in the chemoreceptor region compared with nonchemoreceptor regions. The responses to hypercapnic acidosis were consistent with the hypothesis that CO2 chemoreceptors should exhibit reduced or no pHi recovery in response to CO2-induced cellular acidification just as Ritucci et al. (24) found in rat brain stem slices and Buckler et al. (5, 6) found in isolated type I carotid body cells.
The lack of pHi regulation could be due to the absence of pHi recovery mechanisms in neurons from the chemosensitive area or the inhibition of pHi regulation during hypercapnic acidosis. To investigate whether chemoreceptor region neurons simply lack effective pHi recovery mechanisms, we acidified the neurons while maintaining pHe constant using an ammonia prepulse protocol. The rate of pHi recovery in chemoreceptor region neurons was still slower than recovery in nonchemoreceptor regions, but pHi recovery within the chemoreceptor region was much faster when pHe was equal to the control pHe than recovery during hypercapnic acidosis. Hence, neurons within the chemoreceptor region possess pHi regulatory mechanisms, but the mechanisms were inhibited by hypercapnic acidosis. Therefore, we tried to determine whether the lack of pHi regulation was the result of the hypercapnia or the extracellular acidosis. Regulation of pHi during isohydric hypercapnia (5% CO2) was more rapid than pHi regulation during hypercapnic acidosis (5% CO2), from which we infer that pHe inhibited pHi regulation in neurons within the chemoreceptor region. The pHi recovery mechanisms of a variety of cell types are inhibited by a decrease in pHe (22). Among the conditions we studied, pHi recovery was faster when pHe was held constant. Neurons in the chemoreceptor region had a slower rate of pHi recovery compared with neurons from the nonchemoreceptor regions whether pHe and pHi changed (hypercapnic acidosis) or only pHi changed (ammonia prepulse and isohydric hypercapnia). We infer from the reduced rates of pHi recovery during ammonia prepulse and isohydric hypercapnia that the capacity for pHi regulation was reduced in the chemoreceptor region even at the control pHe (7.8). The rate of pHi recovery was further reduced when pHe was also reduced.pHi regulatory mechanisms in chemoreceptor and
nonchemoreceptor neurons.
We examined the type of pHi regulatory mechanisms present
in neurons within the chemoreceptor region compared with
nonchemoreceptor regions. We used amiloride to inhibit
Na+/H+ exchange and DIDS to inhibit
Cl-dependent HCO3 exchange. The pattern
of inhibition of pHi regulation did not differ
significantly between chemosensitive and nonchemosensitive neurons when
tested with amiloride and/or DIDs during isohydric hypercapnia. Hence,
we found no evidence that different pHi regulatory mechanisms were present in neurons within the chemosensitive region compared with nonchemosensitive regions. When DIDS alone was applied, the rate of pHi recovery during isohydric hypercapnia and
DIDS administration was equivalent to the rate of pHi
recovery when neurons were perfused with an inhibitor-free isohydric
hypercapnic solution. When amiloride was applied, the rate of recovery
was significantly slowed. Thus the dominant pHi regulatory
mechanism in both chemoreceptor and nonchemoreceptor regions seems to
be an Na+/H+ exchanger. However, when both
amiloride and DIDS were applied, the rate of pHi recovery
was further reduced below the rate of recovery during perfusion with
amiloride alone. The data suggest that Cl-dependent
HCO3 exchange may also regulate pHi, but
the Na+/H+ exchanger suffices to regulate
pHi when Cl-dependent HCO3
exchange is inhibited. Thomas (29, 30) put
forward the idea that Na+-dependent
Cl/HCO3 exchange was the essential
pHi-regulating transporter in Helix neurons, but
recently Thomas (31) also found evidence of an Na+/H+ exchange mechanism in Helix
neurons. Hence, it seems likely that pHi regulatory
mechanisms are more heterogeneous in Helix than was first appreciated.
Perspectives
pHi regulation and ventilatory control. In our previous electrophysiological studies of CO2-chemosensitive neurons, we found very few intrinsically CO2-sensitive neurons in each chemosensitive area, perhaps 8-12 neurons (14). It was our expectation that poor pHi regulation might be present only in a few cells in the chemosensitive area of H. aspersa, and we did find that poor pHi regulation among neurons in the subesophageal ganglia was significantly (P = 0.007) segregated and much more likely in neurons confined to the CO2-sensitive area. However, the segregation was not perfect; many neurons outside the CO2-sensitive area demonstrated poor pHi recovery during hypercapnic acidosis. Ritucci et al. (24) also expected only 30-40% of the neurons in chemosensitive regions to show delayed or reduced pHi recovery during hypercapnia, but found poor pHi recovery in the majority of cells in chemosensitive areas. In the NTS, 36 of 39 neurons did not recover; in the VLM, 33 of 38 neurons did not recover. These findings contrast with prompt pHi recovery in 100% of nonchemosensitive neurons in the rat brain stem. The implication of these results is that identification of neurons as chemosensitive based on pHi regulatory profiles will be an insensitive marker of chemosensitivity: poor pHi regulation is ubiquitous, electrophysiological evidence of CO2 chemosensitivity is more circumscribed. We conclude that a delayed or flat pHi recovery profile during intracellular and extra-cellular acidification is a necessary, but not sufficient, condition for CO2 chemoreceptor neurons in both molluscan and murine preparations.
If neurons with flat pHi regulatory profiles during hypercapnic acidosis play an important role in CO2 chemosensory regulation of ventilation (and that is certainly our hypothesis), then the whole animal ventilatory responses to manipulations of pHi should correlate well with the single chemoreceptor neuron response. This is not, however, uniformly the case. Perfusion of the brain stem of awake rabbits with artificial cerebrospinal fluid containing 10 µM DIDS did not change resting ventilation, but did increase the ventilatory response to CO2 (20). These results imply that DIDS reduced the pH in or about chemoreceptor cells. However, DIDS had no effect on pHi or pHi regulation during hypercapnic acidosis in the rat brain stem (24). Similar problems of interpretation exist in snails. DIDS increased normocapnic pneumostomal activity in H. aspersa (10), but DIDS alone did not alter pHi or pHi regulation in neurons within the chemoreceptor area. Amiloride (1 mM) administered via cisternal perfusion to anesthetized rabbits increased minute ventilation under control conditions, but did not alter ventilatory sensitivity to CO2 (21). The effects of amiloride on pHi and pHi regulation in chemoreceptor area neurons in the rat brain stem are the exact opposite of those expected: amiloride did not change pHi under control, normocapnic conditions but did reduce pHi regulation during hypercapnia (24). We have not yet tested the effect of amiloride on pneumostomal activity. The divergence between whole animal ventilatory responses and chemoreceptor area pHi responses is not irreconcilable. For example, the DIDS effects in the whole animal might reflect changes in pHe regulation originating in nonchemoreceptor areas that nonetheless alter pHe in chemoreceptor areas and thereby modify chemoreceptor activity. The responses may also originate from non-acid-base effects of the drugs that obscure the drug effects on pHi. For example, amiloride caused marked generalized excitation in awake rabbits during cisternal perfusion (21). Finally, there is, as yet, no electrophysiological proof that neurons in the NTS and rostral ventrolateral medulla of rats and in the chemosensitive area of Helix, in which pHi regulation is poor, are actually CO2 chemosensors, although that is our working hypothesis. Nonetheless, the lack of correlation between pharmacological manipulation of the whole animal ventilatory responses to CO2 and single-neuron pHi regulation is disconcerting for any theory of respiratory control that posits a key role for pHi in chemoreceptor areas.| |
ACKNOWLEDGEMENTS |
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We gratefully acknowledge the technical assistance of Christopher Welty, a Presidential Scholar at Dartmouth College. Dr. J. S. Erlichman provided invaluable guidance conducting these experiments.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-51238. J. M. Mok was a Presidential Scholar at Dartmouth College.
Address for reprint requests and other correspondence: J. C. Leiter, Dept. of Physiology, Borwell Bldg., Dartmouth Medical School, Lebanon, NH 03756 (E-mail: james.c.leiter{at}dartmouth.edu).
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 19 July 1999; accepted in final form 6 March 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES
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1.
Barnhart, MC.
Control of acid-base status in active and dormant land snails, Otala lactea (Pulmonata, Helicidae).
J Comp Physiol [B]
156:
347-354,
1986.
2.
Benos, D.
Amiloride: a molecular probe of sodium transport in tissues and cells.
Am J Physiol Cell Physiol
242:
C131-C145,
1982
3.
Boron, WF,
and
De Weer P.
Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors.
J Gen Physiol
67:
91-112,
1976
4.
Boyarsky, G,
Ganz MB,
Sterzel RB,
and
Boron WF.
pH regulation in single glomerular mesangial cells. I. Acid extrusion in the absence and presence of HCO3.
Am J Physiol Cell Physiol
255:
C844-C856,
1988
5.
Buckler, KJ,
Vaughan-Jones RD,
Peers C,
Lagadic-Gossman D,
and
Nye PCG
Effects of extracellular pH, PCO2 and HCO3 on intracellular pH in isolated type-I cells of the neonatal rat carotid body.
J Physiol (Lond)
444:
703-721,
1991
6.
Buckler, KJ,
Vaughan-Jones RD,
Peers C,
and
Nye PCG
Intracellular pH and its regulation in isolated type 1 carotid body cells of the neonatal rat.
J Physiol (Lond)
436:
107-129,
1991
7.
Burton, RF.
Buffers in the blood of the snail, Helix pomatia L.
Comp Biochem Physiol A Physiol
29:
919-930,
1969.
8.
Cabantchik, ZI,
and
Greger R.
Chemical probes for anion transporters of mammalian cell membranes.
Am J Physiol Cell Physiol
262:
C803-C827,
1992
9.
Erlichman, JS,
Coates EL,
and
Leiter JC.
Carbonic anhydrase and CO2 chemoreception in the pulmonate snail, Helix aspersa.
Respir Physiol
98:
27-41,
1994[Web of Science][Medline].
10.
Erlichman, JS,
and
Leiter JC.
Central chemoreceptor stimulus in the terrestrial, pulmonate snail, Helix aspersa.
Respir Physiol
95:
209-226,
1994[Web of Science][Medline].
11.
Erlichman, JS,
and
Leiter JC.
CO2 chemoreception in the pulmonate snail, Helix aspersa.
Respir Physiol
93:
347-363,
1993[Web of Science][Medline].
12.
Erlichman, JS,
and
Leiter JC.
Comparative aspects of central CO2 chemoreception.
Respir Physiol
110:
177-185,
1997[Web of Science][Medline].
13.
Erlichman JS and Leiter JC. Identification of central, respiratory
CO2 chemoreceptors in Helix pomatia. Proc
Ann Mtg Soc Integrative Comparative Biol Washington DC 1995.
14.
Erlichman, JS,
and
Leiter JC.
Identification of CO2 chemoreceptors in Helix pomatia.
Am Zool
37:
54-64,
1997.
15.
Loker, J.
The identification of catecholamine and 5-HT containing neurones in the snail brain (PhD thesis). Southhampton, UK: Southhampton University, 1973.
16.
Lu, D,
Erlichman JS,
and
Leiter JC.
Diethyl pyrocarbonate (DEPC) inhibits CO2 chemosensitivity in Helix aspersa.
Respir Physiol
111:
65-78,
1998[Web of Science][Medline].
17.
Millhorn, DE,
and
Eldridge FL.
Role of ventrolateral medulla in regulation of respiratory and cardiovascular systems.
J Appl Physiol
61:
1249-1263,
1986
18.
Nattie, EE.
Central chemoreception.
In: Regulation of Breathing (2nd ed.), edited by Pack AI,
and Dempsey J.. New York: Dekker, 1995, p. 473-510.
19.
Nattie, EE.
Respiratory chemoreception: the acid test.
News Physiol Sci
10:
49-50,
1995
20.
Nattie, EE,
and
Adams JM.
DIDS decreases CSF HCO3 and increased breathing in response to CO2 in awake rabbits.
J Appl Physiol
64:
397-403,
1988
21.
Nattie, EE,
and
Giddings B.
Effects of amiloride and diethyl pyrocarbonate on CSF HCO3 and ventilation in hypercapnia.
J Appl Physiol
65:
242-248,
1988
22.
Putnam, RW.
Intracellular pH regulation.
In: Cell Physiology Source Book, edited by Sperelakis N.. New York: Academic, 1995, p. 212-229.
23.
Ritucci, NA,
Chambers-Kersh L,
Dean JB,
and
Putnam RW.
Intracellular pH regulation in neurons from chemosensitive and nonchemosensitive areas of the medulla.
Am J Physiol Regulatory Integrative Comp Physiol
275:
R1152-R1163,
1998
24.
Ritucci, NA,
Dean JB,
and
Putnam RW.
Intracellular pH responses to hypercapnia in neurons from chemosensitive areas of the medulla.
Am J Physiol Regulatory Integrative Comp Physiol
273:
R433-R441,
1997
25.
Ritucci, NA,
Erlichman JS,
Dean JB,
and
Putnam RW.
A fluorescence technique to measure intracellular pH of single neurons in brainstem slices.
J Neurosci Methods
68:
149-163,
1996[Web of Science][Medline].
26.
Roos, A,
and
Boron WF.
Intracellular pH.
Physiol Rev
61:
296-434,
1981
27.
Thomas, RC.
The effect of carbon dioxide on the intracellular pH and buffering power of snail neurones.
J Physiol (Lond)
255:
715-735,
1976
28.
Thomas, RC.
Intracellular pH of snail neurones measured with a new pH-sensitive glass micro-electrode.
J Physiol (Lond)
238:
159-180,
1974
29.
Thomas, RC.
Ionic mechanism of H+ pump in a snail neurone.
Nature
262:
54-55,
1976[Medline].
30.
Thomas, RC.
The role of bicarbonate, chloride and sodium ions in the regulation of intracellular pH in snail neurones.
J Physiol (Lond)
273:
317-338,
1977
31.
Willoughby, D,
Thomas RC,
and
Schwiening CJ.
A role for Na+/H+ exchange in pH regulation of Helix neurones.
Pflügers Arch
438:
741-749,
1999[Web of Science][Medline].
32.
Winterstein, H.
Chemical control of pulmonary ventilation. III. The "reaction theory" of respiratory control.
N Engl J Med
255:
331-337,
1956.
33.
Winterstein, H.
The "Reaction Theory" of respiratory regulation.
Experientia
5:
221-226,
1949[Medline].
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) and outside the chemoreceptor region
(
). The initial pHi values in each
condition were the lowest pHi values measured within 3 min
of changing the perfusate. The pHi recovery values were
calculated from the initial pHi and the recovery rate in
each cell to estimate the pHi value that would have been
present after 1 h of acidic stress. There were no differences
among the initial pHi values between chemoreceptor and
nonchemoreceptor regions. * pHi recovery rate of
chemoreceptor and nonchemoreceptor regions were significantly lower at
pHe 7.2 compared with pHe 7.5 (P < 0.01). # pHi recovery rates at
both pHe 7.5 and 7.2 were significantly slower in the
chemoreceptor region compared with nonchemoreceptor region neurons
(P < 0.01).
