Sodium/Proton Exchanger 3 in the Medulla Oblongata and Set Point of Breathing Control
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美国呼吸和危急护理医学 2005年第7期
Department of Physiology, University of Duisburg-Essen, Essen
Department of Physiology, Ruhr University, Bochum, Germany
ABSTRACT
Rationale: In vivo inhibition of the sodium/proton exchanger 3 (NHE3) in chemosensitive neurons of the ventrolateral brainstem augments central respiratory drive in anesthetized rabbits. Objectives: To further explore the possible role of this exchanger for the control of breathing, we examined the individual relationship between brainstem NHE3 abundance and ventilation in rabbits during wakefulness. Methods: In 32 adult male rabbits on standard nutritional alkali load, alveolar ventilation, metabolic CO2 production, and blood gases were determined, together with arterial and urinary acid-base status and renal base control functions. Expression of NHE3 in brainstem tissue from the obex region was determined by quantitative real-time reverse-transcription polymerase chain reaction analysis. Measurements and Main Results: Regarding the distribution above and below the median, we classified high and low brainstem NHE3 animals, expressing a mean (± SEM) NHE3 mRNA of 2.08 ± 0.28 and 0.72 ± 0.06 fg cDNA/mg RNA, respectively. Alveolar ventilation of high brainstem NHE3 animals was lower than that of low brainstem NHE3 animals (715 ± 36 vs. 919 ± 41 ml · minuteeC1; p < 0.01), a finding also reflected by a marked difference in PaCO2 (5.24 ± 0.16 vs. 4.44 ± 0.15 kPa; p < 0.01). Among possible secondary factors, CO2 production, systemic base excess, and fractional renal base reabsorption were not found to be different. Conclusions: We conclude that the level of brainstem NHE3 expression—most likely via intracellular pH modulation—contributes to the individual control of breathing and PaCO2 in conscious rabbits by adjusting the set point and the loop gain of the system.
Key Words: alveolar ventilation; arterial PCO2; central chemosensitivity; metabolic rate; renal acid-base control
Alveolar ventilation and PaCO2 show interindividual differences in humans and rabbits (1eC3). The physiologic basis underlying this phenomenon is presently unknown. Former studies have shown that CO2 is the dominant stimulus for central chemosensitive neurons, and that their firing response is at least in part mediated by an increase in intracellular free protons (4eC6). With respect to acid extrusion, neurons within chemosensitive areas of the brainstem make use of sodium/proton exchange, whereas Na+-dependent chloride bicarbonate exchange is apparently not involved (6).
Up to now, eight sodium/proton exchangers have been identified (NHE1eC8), some of which are allosterically activated by free intracellular protons, such that intracellular pH (pHi) determines a level, beyond which transport activity is enhanced when pHi is decreasing (7eC9).
NHE3, whose expression is studied here, was first discovered in kidney (10) and intestine (11). Within proximal tubules of the kidney, NHE3 is profoundly involved in acid-base regulation (12, 13). In the brain, it is found in areas of the ventrolateral brainstem with prevalence for respiration (14eC17). Accordingly, selective inhibitors of the NHE3 applied to CO2/H+-sensitive neurons of the ventrolateral medulla oblongata in vitro lowered the steady-state pHi and increased action potential firing (16, 18). In vivo, a brain-permeant NHE3 inhibitor shifted the hypercapnic respiratory response to the range of smaller PCO2 values and significantly lowered the apneic threshold PCO2 in anesthetized rabbits (17). These findings raised the concept that ventilation could depend on the expression of NHE3, which apparently controls pHi in chemosensitive neurons. This was also suggested by a pilot study in which semiquantitatively determined brainstem NHE3 and PaCO2 were weakly correlated (19). To further test the role of NHE3 as a possible key molecule for breathing control, we now determined alveolar ventilation (A) and used quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) to measure the expression of NHE3 mRNA in the obex region. Possible effects on ventilation originating from metabolic rate, systemic acid-base conditions, or renal base control could be ruled out by our measurements. We were able to show an inverse correlation between ventilation and brainstem NHE3 expression in conscious rabbits. Concerning the relationship between A and PaCO2, the animals matched with the species' normal range for the metabolic hyperbola (3), but those with high and low NHE3 expression were clearly different from each other. Parts of this study have been presented as an abstract (20).
METHODS
Animals
Thirty-two healthy adult male rabbits (Chinchilla Bastard; Charles River, Sulzfeld, Germany) weighing 3.30 ± 0.07 kg were accustomed to a metabolism cage for several days before measurements and kept on species-adapted standard pellet food (Altromin 2123; Altromin GmbH, Lage, Germany) and water ad libitum.
Blood Analysis
Blood samples were taken from the central ear artery under local skin anesthesia (3) to analyze oxygen and CO2 partial pressures and arterial pH by conventional equipment (ABL 5; Radiometer, Copenhagen, Denmark). Base excess and standard bicarbonate concentrations (HCO3eCst) were directly determined by the two-gas equilibration method (3, 17). Creatinine concentrations in serum (and urine) were assayed photometrically with standard test combinations (Labor + Technik; Eberhard Lehmann, Berlin, Germany). Serum proteins were determined by a clinical laboratory.
Assessment of Metabolic Rate and A
The metabolic CO2 production (CO2) was assessed by placing the animals for 25 minutes (10 minutes adaptation, 15 minutes measurement) into a box flooded with room air. The expired CO2 bound to barium hydroxide Ba(OH)2 was determined by differential titration with HCl representing millimoles of CO2. CO2 was converted to ml/minute under standard conditions (STPD). The A under body conditions (BTPS) is then given as (3).
Urine Analysis
As described previously in detail (3), the 24-hour urine was collected under a 30-mm paraffin oil layer to prevent the loss of CO2. The acid-base status of the urine was investigated titrimetrically (pH-electrode and Titrator DL 70 ES; Mettler-Toledo, Gieen, Germany). The fluid and precipitated portions of the urine were analyzed separately for bicarbonate and carbonate, to assess total base excretion (3). The endogenous creatinine clearance was taken to estimate glomerular filtration rate and renal base excretion in relation to glomerular filtration rate. The fractional renal base reabsorption is then given as ratio of absolute reabsorption and filtration (HCO3eCreabs/HCO3eCfil).
RNA Preparation and Quantitative Real-Time RT-PCR
Total RNA was prepared from rabbit medulla oblongata as described previously (17, 21). Tissue was excised by two transversal cuts performed 3.0 ± 0.5 mm caudal and rostral to the obex, respectively, and snap frozen in liquid nitrogen approximately 5 minutes after death. Whole blood samples (5 ml) were investigated in parallel. Quality of isolated RNA was controlled by gel electrophoresis. RNA (1 or 5 e) was reverse transcribed into cDNA with oligodT15 as a primer for reverse transcriptase (AMV reverse transcriptase; Promega, Mannheim, Germany). Qualitative PCRs were performed for NHE3 (Gene Bank Accession No. M85300) to test for the quality of cDNA preparations. Primers used for qualitative and quantitative PCR of NHE3 were as follows: gag gac ata tcc ggg cag at (5' NHE3) and cct tca ggt tca gct cgt gg (3' NHE3); amplicon size was 151 bp. Real-time RT-PCR was performed with SYBR Green (Invitrogen/Molecular Probes, Basel, Switzerland) as fluorescent dye using the Gene Amp 5700 Sequence Detection System (Applied Biosystems, Weiterstadt, Germany). The cDNA standards were prepared from the specific PCR products using a DNA purification kit (Roche, Mannheim, Germany) and adjusted to 0.001 to 100 fg/e by photometric measurement. Quantification of NHE3 mRNA was performed in a two-step real-time PCR with a denaturation step at 95°C (10 minutes) followed by 40 cycles of 95°C (15 seconds) and 60°C (1 minute). All cDNA concentrations from brain samples were normalized to an amount of 1 e of total RNA.
Experimental Protocol
A group of 32 animals was investigated after being accustomed to the laboratory environment. For each animal, the CO2 production was determined at the same time on different days (8:00eC8:30 A.M.). Subsequently, blood samples from the central ear artery were taken under local skin anesthesia, whereby the animals inhaled oxygen-enriched air , to minimize lactate formation and peripheral chemoreflex responses at mean arterial PO2 levels of approximately 28 kPa (3, 17). Comprising the experimental group, 18 animals were killed by an overdose of anesthesia to quickly remove brainstem tissue for RNA preparation. The remaining 14 animals served as a reference group on the same type of food.
Statistical Analysis
Data were averaged to obtain individual and group mean values, SD, and SEM. After corroborating normal distribution (one-sample Kolmogorov-Smirnov test), group mean values were tested for significant differences by independent samples t tests. The limit of significance was at p 0.05. Correlation between selected variables and 95% mean confidence intervals were determined by regression analysis. In addition to correlation analysis, the experimental group was divided into two-percentile subgroups, gathering values above and below the median of 1.0 fg cDNA/e total RNA. Statistical analysis was in part performed by using SPSS 8.0 for Windows software (SPSS, Inc., Chicago, IL).
RESULTS
General Animal Data
Table 1 shows general baseline behavioral data of the investigated rabbits, randomly assigned to an experimental group (n = 18), in which NHE3 mRNA expression was assayed in the brainstem, and to a reference group (n = 14) kept under the same conditions but with undetermined NHE3 expression. There were no significant differences between these groups with respect to food mineral composition, daily food intake, excreted urinary volume, or glomerular filtration rate, except for a slight difference in daily water consumption. Table 1 also provides data of the experimental group after being subdivided according to different brainstem NHE3 expression (see below). Most important, animals of the experimental subgroups were uniform with respect to all balance data.
Quantification of NHE3 mRNA in Rabbit Brainstem Tissue
NHE3 mRNA expression within the obex region was measured by quantitative real-time PCR (Figure 1A). Primers designed for this investigation yielded a highly reliable standard curve (correlation coefficients, 0.97eC0.99), which allowed the quantification of NHE3 mRNA in the range of 10eC3 to 102 fg (Figure 1B). Agarose gel electrophoresis showed that the respective PCR products match the expected size of 151 bp for RNA samples from both brainstem and kidney (Figure 1B, inset), the latter serving as an additional control. Analysis of brainstem samples from 18 rabbits showed that brainstem NHE3 mRNA expression varied considerably between animals. Minimum and maximum values amounted to 0.24 and 3.76 fg cDNA/e total RNA, respectively. These tissue values could not have been influenced by blood constituent, because, using the same PCR protocol, no NHE3 mRNA was detected within rabbit arterial blood samples.
Brainstem NHE3 mRNA Expression and Respiration
Figure 2 shows significant correlation between A and abundance of medullary NHE3 mRNA. When comparing two-percentile subgroups of NHE3 abundance with respect to ventilatory, metabolic, and acid-base variables, Table 2 shows significant differences in baseline ventilation and PaCO2: high NHE3 expression together with low A at high PaCO2, and low NHE3 expression with high A at low PaCO2. On the other hand, no differences could be discerned in CO2 production (CO2), as a measure for metabolic rate, or in the metabolic acid-base status in terms of base excess and weakly dissociated anions from serum proteins. Mean values of the experimental group were not significantly different from those of the reference group.
The steady state relationship between A and PaCO2 is shown in Figure 3A, including individual and mean values from the experimental group and the reference group on the same type of food. The average "metabolic hyperbola" for rabbits during wakefulness (3) is based on nonlinear regression analysis and Bohr's formula, yielding a mean CO2 production (± SEM) of 34.1 ± 0.9 ml · minuteeC1 (STPD). It is obvious that the mean values of rabbits with high and low NHE3 expression are clearly separated from each other, but that they do not deviate from the species' normal range within the 95% confidence interval. The same is true when A is scaled to body weight (Figure 3B). Because the CO2 production strikingly depends on variations of voluntary food intake in the rabbits (Figure 4), it is important to notice that the high and low brainstem NHE3 groups not only sufficiently agree with the expected normal range for rabbits (3) but also do not differ with respect to food intake or metabolic rate.
Brainstem NHE mRNA Expression and Renal Function
The absolute amounts of filtered and reabsorbed renal bicarbonate were slightly (but not significantly) higher in the high brainstem NHE3 expression group with higher values of PaCO2. However, the fractional base reabsorption (in percentage of the filtered amount) was nearly the same (p = 0.66) in the high and low brainstem NHE3 groups. Although there is a clear separation between both groups with respect to PaCO2, neither the level of brainstem NHE3 expression nor that of PaCO2 has any influence on fractional renal bicarbonate reabsorption. Rabbits on herbivore standard chow produce an alkaline urine, whereby the average amount of reabsorbed bicarbonate is in the reported range of 83 to 85% (Table 2), but strikingly depends on dietary alkali content and voluntary daily food intake (3). Therefore, it is important that the high and low NHE3 groups do not differ with respect to daily food intake or dietary alkali load (Table 1).
DISCUSSION
NHE3 mRNA Expression and Baseline Ventilation in Conscious Rabbits
This is the first time that a significant inverse correlation between A and brainstem NHE3 mRNA expression is shown in conscious rabbits. Because measurements in these animals were not disturbed by the use of anesthetics and/or invasive experimental instrumentation, we assume that part of the discerned ventilatory variability could be attributed to the level of brainstem NHE3 expression. To further quantify the relationship between A and PaCO2 ("metabolic hyperbola"), the normal range and 95% confidence interval were established by regression analysis for a large number of rabbits during wakefulness (3), wherein the subgroups with high and low NHE3 expression matched very well, but were clearly separated from each other: high expression together with low ventilation (and high PaCO2) and vice versa.
These findings suggest the level of brainstem NHE3 expression to be one possible determinant for the baseline level of ventilation. One may object that the opposite could also be true—namely, that different levels of ventilation and PaCO2 may have influenced the abundance of the NHE3 mRNA in medullary chemosensitive neurons. However, no differences in NHE3 mRNA expression were found between normocapnic control animals and chronically hypercapnic rabbits exposed to 6% CO2 for 72 hours (22). Furthermore, selective NHE3 inhibition apparently increased the ventilatory drive, demonstrated by a reduction in apneic threshold for PaCO2 (17). Together, these findings support the view that ventilation is secondary to NHE3 expression and not vice versa.
Several studies have identified other genes or proteins underlying an inheritable interindividual disparity of the ventilatory response to hypercapnia and hypoxia (23, 24). Breathing responses are, for instance, under the control of NOS-1 (25), endothelin (26), Mash-1 (27), ret-oncogene (28), or muscarinic M3 receptors (29). All these proteins may contribute to the genetic background of our animal collective. However, the significant correlation between A and one single factor—NHE3—further underlines the pivotal role of this ion exchanger for the control of breathing.
Secondary Effects on Baseline Ventilation in Conscious Rabbits
An early study in awake rabbits breathing oxygen-enriched air (2) revealed large interindividual variations in end-tidal PCO2 (up to 15 mm Hg), and in pulmonary ventilation (600eC1500 ml · minuteeC1), clearly indicating a considerable variability of the central respiratory controller. Likewise, interindividual variations of end-tidal and arterialized capillary PCO2 have been described in humans (1), and a great number of possible causal factors have been proposed, such as diurnal changes in vigilance, metabolic rate, acid-base condition, or variations in respiratory and renal control functions.
In our study, diurnal changes in vigilance were rather unlikely, because all measurements were performed at the same time of the day. Animals had been accustomed to laboratory conditions and were devoid of stress symptoms. Regarding the high and low NHE3 groups, there were no differences in metabolic rate because CO2 production and food intake were not different. Accordingly, PaCO2 was not correlated with CO2 (r = eC0.05, p = 0.68, n = 80). In much the same way, there were no group differences in metabolic acid-base conditions, as reflected by base excess or by the concentration of nonbicarbonate buffers (e.g., weakly dissociated serum albumins), that could have affected the relationship between PaCO2 and pH (30). A possible contribution of peripheral chemoreflex control can be excluded as well, because the animals inhaled oxygen-enriched air, such that arterial PO2 values were elevated to levels of approximately 28 kPa, at which arterial chemoreceptors in the rabbit are completely silenced (17).
NHE3 mRNA Expression and Renal Acid-Base Control
Faced with the variability of spontaneous PCO2 levels, Crosby and Robbins (1) suggested an interdependent action of renal and respiratory controllers for the adjustment of PaCO2 and pH. This may be of special importance with respect to NHE3, as this subtype is the main sodium/proton exchanger of the proximal tubule and fundamentally involved in acid-base regulation by the kidney (12, 13). Although further studies are needed to clarify whether NHE3 mRNA expression is equivalently regulated in brainstem and kidney, it must be underlined that herbivore alkali load as well as fractional renal base reabsorption were not different for the groups with high and low brainstem NHE3 levels (Tables 1 and 2). The marked reduction in fractional base reabsorption occurring in rabbits on high-alkali food (3) may reflect adaptive changes in renal NHE3 protein abundance as shown for rats (31). On the other hand, the differences in PaCO2 we found among high and low brainstem NHE3 expression groups were not accompanied by differences in fractional renal base reabsorption. This is in accordance with studies on prolonged respiratory acidosis/alkalosis, revealing no adaptation of the exchanger's abundance (32eC34). Together, changes in ventilation observed here are certainly not secondary to nonrespiratory acid-base changes by renal control.
Tentative Role of Brainstem NHE3 for Central Respiratory Chemosensitivity
Up to now, we have no direct evidence that the level of NHE3 mRNA in the brainstem reflects NHE3 protein abundance, although this is likely from studies on other tissues. We are also aware that NHE3 is a highly regulated antiporter (7, 9), which means that, even at equal concentrations of NHE3 mRNA or protein, differences in activity might be present at the cellular level (32eC34). However, as a measure for all cells within a certain area of the brainstem, it is likely that elevated mRNA levels reflect an overall elevated activity. In line with this, it has to be mentioned that respiration under the influence of a low NHE3 mRNA expression appears analogous to the reduction in apneic threshold PaCO2 that was obtained by selective NHE3 inhibition in anesthetized and artificially ventilated rabbits (17, 22).
Our results further highlight the possible role of NHE3 as a key molecule of pHi regulation within neurons involved in central respiratory chemosensitivity. Although the underlying ultrastructural and molecular elements of the transduction cascade are still unknown, there is convincing evidence from studies in vitro and in vivo that the pHi of chemosensitive neurones, which seems to be under the control of NHE3 (16eC20), is the adequate signal to elicit respiratory responses (4eC6, 16, 18, 35). Concerning the neuronal sensing mechanism, several pHi-sensitive potassium channels, such as TASK-1 (36), several Kir channels (37), or Ca2+-dependent K channels (38), may link up membrane potential of chemosensitive neurons to pHi and may thus serve as pHi sensors. However, the concept of one single channel acting as a pHi sensor is complicated by the fact that CO2/H+-sensitive neurons do not necessarily depolarize on hypercapnia (18) and that CO2 responses of these neurons are sensitive to intracellular dialysis (39), suggesting the occurrence of further cytoplasmic factors.
Pathophysiologic Implications of Brainstem NHE3 mRNA Expression
On the basis of control theory of breathing (40), the overall gain of a feedback loop is given as the ratio of the controller gain and the "plant gain" of the controlled system (41eC44). At constant CO2 production and/or CO2 delivery by lung perfusion, the plant gain is in the inverse ratio of the squared set point PCO2. Consequently, a lowered set point PCO2 is a factor to reduce the loop gain and this, connected with a larger difference between eupneic and apneic PCO2, is generally expected to prevent ventilatory instability and variability (42eC44). A genetically determined overexpression of NHE3 in central chemosensitive areas leading to lower ventilation with high values of PCO2 may therefore predispose individuals to central apnea and sleep-disordered breathing. Interestingly, the activity of overall sodium/proton exchange in human blood cells has been suggested as an indicator of the ventilatory CO2 sensitivity, because high NHE activity hinted at a dangerous CO2 retainment in "nonresponders" during CO2 exposure (45). In line with these observations, a clinical study showed increased sodium/proton exchanger activity of lymphocytes in patients prone to sleep apnea (46). It is tempting to speculate that brain-permeant NHE3 inhibitors may be protective against sleep-disordered breathing, at least under conditions of hypercapnia but normal lung perfusion (47).
Together, our findings support the hypothesis that the NHE3 within the brainstem is involved in central breathing control. More specifically, its differential expression may explain in part the interindividual variation of baseline ventilation and set point PCO2 in conscious rabbits.
Acknowledgments
The authors thank Dr. Hermann Kalhoff (Pediatric Clinic, Dortmund), for providing laboratory facilities, and Patricia Freitag, for her skillful technical assistance.
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Department of Physiology, Ruhr University, Bochum, Germany
ABSTRACT
Rationale: In vivo inhibition of the sodium/proton exchanger 3 (NHE3) in chemosensitive neurons of the ventrolateral brainstem augments central respiratory drive in anesthetized rabbits. Objectives: To further explore the possible role of this exchanger for the control of breathing, we examined the individual relationship between brainstem NHE3 abundance and ventilation in rabbits during wakefulness. Methods: In 32 adult male rabbits on standard nutritional alkali load, alveolar ventilation, metabolic CO2 production, and blood gases were determined, together with arterial and urinary acid-base status and renal base control functions. Expression of NHE3 in brainstem tissue from the obex region was determined by quantitative real-time reverse-transcription polymerase chain reaction analysis. Measurements and Main Results: Regarding the distribution above and below the median, we classified high and low brainstem NHE3 animals, expressing a mean (± SEM) NHE3 mRNA of 2.08 ± 0.28 and 0.72 ± 0.06 fg cDNA/mg RNA, respectively. Alveolar ventilation of high brainstem NHE3 animals was lower than that of low brainstem NHE3 animals (715 ± 36 vs. 919 ± 41 ml · minuteeC1; p < 0.01), a finding also reflected by a marked difference in PaCO2 (5.24 ± 0.16 vs. 4.44 ± 0.15 kPa; p < 0.01). Among possible secondary factors, CO2 production, systemic base excess, and fractional renal base reabsorption were not found to be different. Conclusions: We conclude that the level of brainstem NHE3 expression—most likely via intracellular pH modulation—contributes to the individual control of breathing and PaCO2 in conscious rabbits by adjusting the set point and the loop gain of the system.
Key Words: alveolar ventilation; arterial PCO2; central chemosensitivity; metabolic rate; renal acid-base control
Alveolar ventilation and PaCO2 show interindividual differences in humans and rabbits (1eC3). The physiologic basis underlying this phenomenon is presently unknown. Former studies have shown that CO2 is the dominant stimulus for central chemosensitive neurons, and that their firing response is at least in part mediated by an increase in intracellular free protons (4eC6). With respect to acid extrusion, neurons within chemosensitive areas of the brainstem make use of sodium/proton exchange, whereas Na+-dependent chloride bicarbonate exchange is apparently not involved (6).
Up to now, eight sodium/proton exchangers have been identified (NHE1eC8), some of which are allosterically activated by free intracellular protons, such that intracellular pH (pHi) determines a level, beyond which transport activity is enhanced when pHi is decreasing (7eC9).
NHE3, whose expression is studied here, was first discovered in kidney (10) and intestine (11). Within proximal tubules of the kidney, NHE3 is profoundly involved in acid-base regulation (12, 13). In the brain, it is found in areas of the ventrolateral brainstem with prevalence for respiration (14eC17). Accordingly, selective inhibitors of the NHE3 applied to CO2/H+-sensitive neurons of the ventrolateral medulla oblongata in vitro lowered the steady-state pHi and increased action potential firing (16, 18). In vivo, a brain-permeant NHE3 inhibitor shifted the hypercapnic respiratory response to the range of smaller PCO2 values and significantly lowered the apneic threshold PCO2 in anesthetized rabbits (17). These findings raised the concept that ventilation could depend on the expression of NHE3, which apparently controls pHi in chemosensitive neurons. This was also suggested by a pilot study in which semiquantitatively determined brainstem NHE3 and PaCO2 were weakly correlated (19). To further test the role of NHE3 as a possible key molecule for breathing control, we now determined alveolar ventilation (A) and used quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) to measure the expression of NHE3 mRNA in the obex region. Possible effects on ventilation originating from metabolic rate, systemic acid-base conditions, or renal base control could be ruled out by our measurements. We were able to show an inverse correlation between ventilation and brainstem NHE3 expression in conscious rabbits. Concerning the relationship between A and PaCO2, the animals matched with the species' normal range for the metabolic hyperbola (3), but those with high and low NHE3 expression were clearly different from each other. Parts of this study have been presented as an abstract (20).
METHODS
Animals
Thirty-two healthy adult male rabbits (Chinchilla Bastard; Charles River, Sulzfeld, Germany) weighing 3.30 ± 0.07 kg were accustomed to a metabolism cage for several days before measurements and kept on species-adapted standard pellet food (Altromin 2123; Altromin GmbH, Lage, Germany) and water ad libitum.
Blood Analysis
Blood samples were taken from the central ear artery under local skin anesthesia (3) to analyze oxygen and CO2 partial pressures and arterial pH by conventional equipment (ABL 5; Radiometer, Copenhagen, Denmark). Base excess and standard bicarbonate concentrations (HCO3eCst) were directly determined by the two-gas equilibration method (3, 17). Creatinine concentrations in serum (and urine) were assayed photometrically with standard test combinations (Labor + Technik; Eberhard Lehmann, Berlin, Germany). Serum proteins were determined by a clinical laboratory.
Assessment of Metabolic Rate and A
The metabolic CO2 production (CO2) was assessed by placing the animals for 25 minutes (10 minutes adaptation, 15 minutes measurement) into a box flooded with room air. The expired CO2 bound to barium hydroxide Ba(OH)2 was determined by differential titration with HCl representing millimoles of CO2. CO2 was converted to ml/minute under standard conditions (STPD). The A under body conditions (BTPS) is then given as (3).
Urine Analysis
As described previously in detail (3), the 24-hour urine was collected under a 30-mm paraffin oil layer to prevent the loss of CO2. The acid-base status of the urine was investigated titrimetrically (pH-electrode and Titrator DL 70 ES; Mettler-Toledo, Gieen, Germany). The fluid and precipitated portions of the urine were analyzed separately for bicarbonate and carbonate, to assess total base excretion (3). The endogenous creatinine clearance was taken to estimate glomerular filtration rate and renal base excretion in relation to glomerular filtration rate. The fractional renal base reabsorption is then given as ratio of absolute reabsorption and filtration (HCO3eCreabs/HCO3eCfil).
RNA Preparation and Quantitative Real-Time RT-PCR
Total RNA was prepared from rabbit medulla oblongata as described previously (17, 21). Tissue was excised by two transversal cuts performed 3.0 ± 0.5 mm caudal and rostral to the obex, respectively, and snap frozen in liquid nitrogen approximately 5 minutes after death. Whole blood samples (5 ml) were investigated in parallel. Quality of isolated RNA was controlled by gel electrophoresis. RNA (1 or 5 e) was reverse transcribed into cDNA with oligodT15 as a primer for reverse transcriptase (AMV reverse transcriptase; Promega, Mannheim, Germany). Qualitative PCRs were performed for NHE3 (Gene Bank Accession No. M85300) to test for the quality of cDNA preparations. Primers used for qualitative and quantitative PCR of NHE3 were as follows: gag gac ata tcc ggg cag at (5' NHE3) and cct tca ggt tca gct cgt gg (3' NHE3); amplicon size was 151 bp. Real-time RT-PCR was performed with SYBR Green (Invitrogen/Molecular Probes, Basel, Switzerland) as fluorescent dye using the Gene Amp 5700 Sequence Detection System (Applied Biosystems, Weiterstadt, Germany). The cDNA standards were prepared from the specific PCR products using a DNA purification kit (Roche, Mannheim, Germany) and adjusted to 0.001 to 100 fg/e by photometric measurement. Quantification of NHE3 mRNA was performed in a two-step real-time PCR with a denaturation step at 95°C (10 minutes) followed by 40 cycles of 95°C (15 seconds) and 60°C (1 minute). All cDNA concentrations from brain samples were normalized to an amount of 1 e of total RNA.
Experimental Protocol
A group of 32 animals was investigated after being accustomed to the laboratory environment. For each animal, the CO2 production was determined at the same time on different days (8:00eC8:30 A.M.). Subsequently, blood samples from the central ear artery were taken under local skin anesthesia, whereby the animals inhaled oxygen-enriched air , to minimize lactate formation and peripheral chemoreflex responses at mean arterial PO2 levels of approximately 28 kPa (3, 17). Comprising the experimental group, 18 animals were killed by an overdose of anesthesia to quickly remove brainstem tissue for RNA preparation. The remaining 14 animals served as a reference group on the same type of food.
Statistical Analysis
Data were averaged to obtain individual and group mean values, SD, and SEM. After corroborating normal distribution (one-sample Kolmogorov-Smirnov test), group mean values were tested for significant differences by independent samples t tests. The limit of significance was at p 0.05. Correlation between selected variables and 95% mean confidence intervals were determined by regression analysis. In addition to correlation analysis, the experimental group was divided into two-percentile subgroups, gathering values above and below the median of 1.0 fg cDNA/e total RNA. Statistical analysis was in part performed by using SPSS 8.0 for Windows software (SPSS, Inc., Chicago, IL).
RESULTS
General Animal Data
Table 1 shows general baseline behavioral data of the investigated rabbits, randomly assigned to an experimental group (n = 18), in which NHE3 mRNA expression was assayed in the brainstem, and to a reference group (n = 14) kept under the same conditions but with undetermined NHE3 expression. There were no significant differences between these groups with respect to food mineral composition, daily food intake, excreted urinary volume, or glomerular filtration rate, except for a slight difference in daily water consumption. Table 1 also provides data of the experimental group after being subdivided according to different brainstem NHE3 expression (see below). Most important, animals of the experimental subgroups were uniform with respect to all balance data.
Quantification of NHE3 mRNA in Rabbit Brainstem Tissue
NHE3 mRNA expression within the obex region was measured by quantitative real-time PCR (Figure 1A). Primers designed for this investigation yielded a highly reliable standard curve (correlation coefficients, 0.97eC0.99), which allowed the quantification of NHE3 mRNA in the range of 10eC3 to 102 fg (Figure 1B). Agarose gel electrophoresis showed that the respective PCR products match the expected size of 151 bp for RNA samples from both brainstem and kidney (Figure 1B, inset), the latter serving as an additional control. Analysis of brainstem samples from 18 rabbits showed that brainstem NHE3 mRNA expression varied considerably between animals. Minimum and maximum values amounted to 0.24 and 3.76 fg cDNA/e total RNA, respectively. These tissue values could not have been influenced by blood constituent, because, using the same PCR protocol, no NHE3 mRNA was detected within rabbit arterial blood samples.
Brainstem NHE3 mRNA Expression and Respiration
Figure 2 shows significant correlation between A and abundance of medullary NHE3 mRNA. When comparing two-percentile subgroups of NHE3 abundance with respect to ventilatory, metabolic, and acid-base variables, Table 2 shows significant differences in baseline ventilation and PaCO2: high NHE3 expression together with low A at high PaCO2, and low NHE3 expression with high A at low PaCO2. On the other hand, no differences could be discerned in CO2 production (CO2), as a measure for metabolic rate, or in the metabolic acid-base status in terms of base excess and weakly dissociated anions from serum proteins. Mean values of the experimental group were not significantly different from those of the reference group.
The steady state relationship between A and PaCO2 is shown in Figure 3A, including individual and mean values from the experimental group and the reference group on the same type of food. The average "metabolic hyperbola" for rabbits during wakefulness (3) is based on nonlinear regression analysis and Bohr's formula, yielding a mean CO2 production (± SEM) of 34.1 ± 0.9 ml · minuteeC1 (STPD). It is obvious that the mean values of rabbits with high and low NHE3 expression are clearly separated from each other, but that they do not deviate from the species' normal range within the 95% confidence interval. The same is true when A is scaled to body weight (Figure 3B). Because the CO2 production strikingly depends on variations of voluntary food intake in the rabbits (Figure 4), it is important to notice that the high and low brainstem NHE3 groups not only sufficiently agree with the expected normal range for rabbits (3) but also do not differ with respect to food intake or metabolic rate.
Brainstem NHE mRNA Expression and Renal Function
The absolute amounts of filtered and reabsorbed renal bicarbonate were slightly (but not significantly) higher in the high brainstem NHE3 expression group with higher values of PaCO2. However, the fractional base reabsorption (in percentage of the filtered amount) was nearly the same (p = 0.66) in the high and low brainstem NHE3 groups. Although there is a clear separation between both groups with respect to PaCO2, neither the level of brainstem NHE3 expression nor that of PaCO2 has any influence on fractional renal bicarbonate reabsorption. Rabbits on herbivore standard chow produce an alkaline urine, whereby the average amount of reabsorbed bicarbonate is in the reported range of 83 to 85% (Table 2), but strikingly depends on dietary alkali content and voluntary daily food intake (3). Therefore, it is important that the high and low NHE3 groups do not differ with respect to daily food intake or dietary alkali load (Table 1).
DISCUSSION
NHE3 mRNA Expression and Baseline Ventilation in Conscious Rabbits
This is the first time that a significant inverse correlation between A and brainstem NHE3 mRNA expression is shown in conscious rabbits. Because measurements in these animals were not disturbed by the use of anesthetics and/or invasive experimental instrumentation, we assume that part of the discerned ventilatory variability could be attributed to the level of brainstem NHE3 expression. To further quantify the relationship between A and PaCO2 ("metabolic hyperbola"), the normal range and 95% confidence interval were established by regression analysis for a large number of rabbits during wakefulness (3), wherein the subgroups with high and low NHE3 expression matched very well, but were clearly separated from each other: high expression together with low ventilation (and high PaCO2) and vice versa.
These findings suggest the level of brainstem NHE3 expression to be one possible determinant for the baseline level of ventilation. One may object that the opposite could also be true—namely, that different levels of ventilation and PaCO2 may have influenced the abundance of the NHE3 mRNA in medullary chemosensitive neurons. However, no differences in NHE3 mRNA expression were found between normocapnic control animals and chronically hypercapnic rabbits exposed to 6% CO2 for 72 hours (22). Furthermore, selective NHE3 inhibition apparently increased the ventilatory drive, demonstrated by a reduction in apneic threshold for PaCO2 (17). Together, these findings support the view that ventilation is secondary to NHE3 expression and not vice versa.
Several studies have identified other genes or proteins underlying an inheritable interindividual disparity of the ventilatory response to hypercapnia and hypoxia (23, 24). Breathing responses are, for instance, under the control of NOS-1 (25), endothelin (26), Mash-1 (27), ret-oncogene (28), or muscarinic M3 receptors (29). All these proteins may contribute to the genetic background of our animal collective. However, the significant correlation between A and one single factor—NHE3—further underlines the pivotal role of this ion exchanger for the control of breathing.
Secondary Effects on Baseline Ventilation in Conscious Rabbits
An early study in awake rabbits breathing oxygen-enriched air (2) revealed large interindividual variations in end-tidal PCO2 (up to 15 mm Hg), and in pulmonary ventilation (600eC1500 ml · minuteeC1), clearly indicating a considerable variability of the central respiratory controller. Likewise, interindividual variations of end-tidal and arterialized capillary PCO2 have been described in humans (1), and a great number of possible causal factors have been proposed, such as diurnal changes in vigilance, metabolic rate, acid-base condition, or variations in respiratory and renal control functions.
In our study, diurnal changes in vigilance were rather unlikely, because all measurements were performed at the same time of the day. Animals had been accustomed to laboratory conditions and were devoid of stress symptoms. Regarding the high and low NHE3 groups, there were no differences in metabolic rate because CO2 production and food intake were not different. Accordingly, PaCO2 was not correlated with CO2 (r = eC0.05, p = 0.68, n = 80). In much the same way, there were no group differences in metabolic acid-base conditions, as reflected by base excess or by the concentration of nonbicarbonate buffers (e.g., weakly dissociated serum albumins), that could have affected the relationship between PaCO2 and pH (30). A possible contribution of peripheral chemoreflex control can be excluded as well, because the animals inhaled oxygen-enriched air, such that arterial PO2 values were elevated to levels of approximately 28 kPa, at which arterial chemoreceptors in the rabbit are completely silenced (17).
NHE3 mRNA Expression and Renal Acid-Base Control
Faced with the variability of spontaneous PCO2 levels, Crosby and Robbins (1) suggested an interdependent action of renal and respiratory controllers for the adjustment of PaCO2 and pH. This may be of special importance with respect to NHE3, as this subtype is the main sodium/proton exchanger of the proximal tubule and fundamentally involved in acid-base regulation by the kidney (12, 13). Although further studies are needed to clarify whether NHE3 mRNA expression is equivalently regulated in brainstem and kidney, it must be underlined that herbivore alkali load as well as fractional renal base reabsorption were not different for the groups with high and low brainstem NHE3 levels (Tables 1 and 2). The marked reduction in fractional base reabsorption occurring in rabbits on high-alkali food (3) may reflect adaptive changes in renal NHE3 protein abundance as shown for rats (31). On the other hand, the differences in PaCO2 we found among high and low brainstem NHE3 expression groups were not accompanied by differences in fractional renal base reabsorption. This is in accordance with studies on prolonged respiratory acidosis/alkalosis, revealing no adaptation of the exchanger's abundance (32eC34). Together, changes in ventilation observed here are certainly not secondary to nonrespiratory acid-base changes by renal control.
Tentative Role of Brainstem NHE3 for Central Respiratory Chemosensitivity
Up to now, we have no direct evidence that the level of NHE3 mRNA in the brainstem reflects NHE3 protein abundance, although this is likely from studies on other tissues. We are also aware that NHE3 is a highly regulated antiporter (7, 9), which means that, even at equal concentrations of NHE3 mRNA or protein, differences in activity might be present at the cellular level (32eC34). However, as a measure for all cells within a certain area of the brainstem, it is likely that elevated mRNA levels reflect an overall elevated activity. In line with this, it has to be mentioned that respiration under the influence of a low NHE3 mRNA expression appears analogous to the reduction in apneic threshold PaCO2 that was obtained by selective NHE3 inhibition in anesthetized and artificially ventilated rabbits (17, 22).
Our results further highlight the possible role of NHE3 as a key molecule of pHi regulation within neurons involved in central respiratory chemosensitivity. Although the underlying ultrastructural and molecular elements of the transduction cascade are still unknown, there is convincing evidence from studies in vitro and in vivo that the pHi of chemosensitive neurones, which seems to be under the control of NHE3 (16eC20), is the adequate signal to elicit respiratory responses (4eC6, 16, 18, 35). Concerning the neuronal sensing mechanism, several pHi-sensitive potassium channels, such as TASK-1 (36), several Kir channels (37), or Ca2+-dependent K channels (38), may link up membrane potential of chemosensitive neurons to pHi and may thus serve as pHi sensors. However, the concept of one single channel acting as a pHi sensor is complicated by the fact that CO2/H+-sensitive neurons do not necessarily depolarize on hypercapnia (18) and that CO2 responses of these neurons are sensitive to intracellular dialysis (39), suggesting the occurrence of further cytoplasmic factors.
Pathophysiologic Implications of Brainstem NHE3 mRNA Expression
On the basis of control theory of breathing (40), the overall gain of a feedback loop is given as the ratio of the controller gain and the "plant gain" of the controlled system (41eC44). At constant CO2 production and/or CO2 delivery by lung perfusion, the plant gain is in the inverse ratio of the squared set point PCO2. Consequently, a lowered set point PCO2 is a factor to reduce the loop gain and this, connected with a larger difference between eupneic and apneic PCO2, is generally expected to prevent ventilatory instability and variability (42eC44). A genetically determined overexpression of NHE3 in central chemosensitive areas leading to lower ventilation with high values of PCO2 may therefore predispose individuals to central apnea and sleep-disordered breathing. Interestingly, the activity of overall sodium/proton exchange in human blood cells has been suggested as an indicator of the ventilatory CO2 sensitivity, because high NHE activity hinted at a dangerous CO2 retainment in "nonresponders" during CO2 exposure (45). In line with these observations, a clinical study showed increased sodium/proton exchanger activity of lymphocytes in patients prone to sleep apnea (46). It is tempting to speculate that brain-permeant NHE3 inhibitors may be protective against sleep-disordered breathing, at least under conditions of hypercapnia but normal lung perfusion (47).
Together, our findings support the hypothesis that the NHE3 within the brainstem is involved in central breathing control. More specifically, its differential expression may explain in part the interindividual variation of baseline ventilation and set point PCO2 in conscious rabbits.
Acknowledgments
The authors thank Dr. Hermann Kalhoff (Pediatric Clinic, Dortmund), for providing laboratory facilities, and Patricia Freitag, for her skillful technical assistance.
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