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Published ahead of print on June 28, 2007, doi:10.1164/rccm.200703-347OC
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American Journal of Respiratory and Critical Care Medicine Vol 176. pp. 513-519, (2007)
© 2007 American Thoracic Society
doi: 10.1164/rccm.200703-347OC

Original Article

Role of Brainstem Sodium/Proton Exchanger 3 for Breathing Control during Chronic Acid–Base Imbalance

Heidrun Kiwull-Schöne1, Peter Kiwull1, Stilla Frede2 and Martin Wiemann2

1 Department of Physiology, Ruhr-University, Bochum, Germany; and 2 Department of Physiology, University of Duisburg-Essen, Essen, Germany

Correspondence and requests for reprints should be addressed to Heidrun Kiwull-Schöne, M.D., Department of Physiology, Ruhr-University, 44780 Bochum, Germany. E-mail: heidrun.kiwull-schoene{at}rub.de


   ABSTRACT
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 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: The sodium/proton exchanger (NHE) 3 is expressed in brainstem areas with prevalence for central chemosensitivity. Selective NHE3 inhibitors can evoke CO2 mimetic responses both in vitro and in vivo, demonstrating the functional significance of this pH-regulating protein. Moreover, levels of NHE3 expression are inversely correlated to interindividual differences of baseline ventilation in conscious rabbits.

Objectives: We explored the influence of chronic acid–base disturbances on mRNA levels of brainstem NHE3 in relation to breathing control.

Methods: Alveolar ventilation (VA), blood gases, systemic base excess (BE), and metabolic VCO2 were determined in rabbits shortly after exposure to either CO2-enriched air for 3 days (n = 5) or to ammonium chloride with drinking water for 2 days (n = 6). Untreated animals served as controls (n = 24). NHE3 mRNA within the obex region was quantified by real-time reverse transcription–polymerase chain reaction.

Measurements and Main Results: After chronic hypercapnia, we found a compensatory rise of BE (mean ± SEM) to 5.3 ± 0.5 mmol · L–1 with slightly elevated PaCO2. Brainstem NHE3 mRNA as well as VA were not significantly different from control levels. In the NH4Cl group, arterial pH was ~0.09 units lower than control, and BE decreased to –6.5 ± 1.6 mmol · L–1 with slightly decreased PaCO2, but considerably reduced VA (by ~25%; P < 0.05) and VCO2. Concomitantly, brainstem NHE3 mRNA had increased from control level of 1.45 ± 0.19 to 3.64 ± 0.37 fg cDNA/µg RNA; P < 0.01.

Conclusions: Expression of brainstem NHE3 is up-regulated by chronic metabolic acidosis but not by prolonged hypercapnia. It is proposed that elevated brainstem NHE3 expression contributes to limit maladaptive hyperventilation during metabolic acidosis.

Key Words: brainstem sodium/proton exchange 3 • central chemosensitivity • chronic metabolic acidosis • prolonged hypercapnia • metabolic rate



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Selective sodium/proton exchanger (NHE3) inhibition in chemosensitive brainstem neurons evokes CO2 mimetic responses. Moreover, individual differences of NHE3 expression correlates to baseline ventilation, demonstrating functional significance of NHE3 for breathing control.

What This Study Adds to the Field
NHE3 mRNA expression in rabbits is significantly up-regulated during chronic metabolic but not respiratory acidosis. Elevated brainstem NHE3 expression likely contributes to limit maladaptive hyperventilation during metabolic acidosis.

 
It is widely accepted 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 (1–3). With respect to acid extrusion, neurons within chemosensitive areas of the brainstem make use of sodium/proton exchange (NHE), whereas bicarbonate-dependent processes appear to play a minor role (3). Up to now, nine subtypes of sodium hydrogen exchangers have been identified (NHE1–9), which accomplish an electroneutral exchange of Na+ for H+. Most of them are highly regulated (glyco)phosphoproteins activated by free intracellular protons (4). The NHE3 is expressed in areas of the ventrolateral brainstem with prevalence for respiration in rats (5, 6) and in rabbits (7). In ventrolateral neurons of organotypic cultures derived from the medulla oblongata, selective NHE3 inhibitors lowered the intracellular pH (pHi). Accordingly, these inhibitors increased action potential firing of CO2/H+-sensitive neurons (6, 8). In vivo, a brain-permeant NHE3 inhibitor shifted the hypercapnic respiratory response to the range of smaller PCO2 values and lowered the arterial threshold PCO2 for central apnea in anaesthetized rabbits (7). Recently, the level of NHE3 mRNA was found to be inversely related to individual levels of baseline ventilation (9). Together, these findings strongly suggest that NHE3 is important for the control of respiration.

On the other hand, NHE3 is the dominant NHE subtype in kidney proximal tubules, where it substantially contributes to the reuptake of Na+ in exchange for H+. Mice deficient for this NHE isotype showed—among other disturbances—a reduced ability of proximal bicarbonate reabsorption (10, 11). Due to the pivotal role NHE3 plays in metabolic acid–base balance, it is not surprising that its abundance and activity is feedback regulated: while alkali load lowered NHE3 protein in proximal tubules (12), NH4Cl-induced acidosis up-regulated it in renal brush border membranes (13, 14). In contrast, prolonged respiratory acidosis had no influence on NHE3 expression in the kidney (15–17), nor did it modify effects of NHE3 inhibitors on central respiratory chemosensitivity (18).

These findings raise the question as to whether chronic metabolic acid–base disturbances can also change NHE3 expression in the brainstem. To test this possibility, we quantified NHE3 mRNA levels in the obex region by real-time reverse transcription–polymerase chain reaction (RT-PCR) in animals subjected either to prolonged hypercapnia or to chronic metabolic acidosis. Because the latter treatment increased NHE3 mRNA levels but decreased ventilation, we speculate that the brainstem NHE3 expression level comprises a meaningful adaptation to physiologic requirements. Based on our measurements of ventilation and acid–base conditions, a model is proposed in which NHE3 overexpression is among factors limiting maladaptive hyperventilation during metabolic acidosis. Parts of this study have been published as an abstract (19).


   METHODS
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 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
A total of 35 healthy adult male rabbits (Chinchilla Bastard; Charles River Laboratories, Inc., Wilmington, MA) were acclimated to a metabolic cage under control conditions and kept on species-adapted standard pellet food (Altromin 2123; Altromin GmbH, Lage, Germany) and water ad libitum. The principles of laboratory animal care of the German Law on the Protection of Animals were followed.

Blood and Urine Analyses and Assessment of Metabolic Rate
As described previously (20), arterial blood gases and pH were measured by conventional equipment (ABL 5; Radiometer, Copenhagen, Denmark). Standard base excess (BE) was determined directly. Lactate and creatinine concentrations in serum (and urine) were assayed photometrically with standard tests (Labor + Technik; Eberhard Lehmann, Berlin, Germany). To assess total base excretion, fluid and precipitated portions of the 24-hour urine were analyzed for bicarbonate and carbonate by titration (pH electrode and Titrator DL 70 ES; Mettler-Toledo, Gießen, Germany). The endogenous creatinine clearance was taken as an estimate of the glomerular filtration rate (GFR).

The metabolic VCO2 was determined by differential titration of expired CO2 bound to barium hydroxide and converted to standard conditions. Alveolar ventilation (VA) under body conditions is then given as VABTPS = VCO2STPD · 863/PaCO2.

RNA Preparation and Quantitative Real-Time RT-PCR
To quantify individual brainstem NHE3 concentrations in coordination with respiratory variables, the transcript NHE3 mRNA was determined by real-time RT-PCR from rabbit medulla oblongata essentially as previously described (7, 9). As such, tissue was excised by two transverse cuts 3.0 (±0.5) mm caudal and rostral to the obex. Tissue from kidney cortex was processed in the same manner. All cDNA concentrations from tissue samples were normalized to an amount of 1 µg of total RNA.

Experimental Protocol
The animals' metabolic CO2 production was determined at the same time on different days (8:00–8:30 A.M.) and blood samples were taken from the central ear artery under local skin anesthesia. Oxygen-enriched air (FIO2 0.4–0.6) was inhaled, to minimize lactate formation and arterial chemoreflex responses (20). Thereby, the average (±SEM) PaO2 and lactate concentration came to 27.2 ± 1.9 kPa (~205 mm Hg) and 2.0 ± 0.3 mmol · L–1, respectively. These procedures were performed under control conditions before and after prolonged hypercapnia (n = 5) or chronic metabolic acidosis (n = 6).

Chronic hypercapnia was achieved by keeping animals individually for 72 hours in a modified semiclosed metabolic cage, constantly flooded with a constant CO2 fraction in air (FCO2 = 0.06). Measurements were performed 30 minutes after return to normal air breathing, at which time eucapnic steady-state conditions had been reached (21). After the measurements (10–15 min), hypercapnia was reestablished for 2–3 hours before animals were killed and tissue samples were taken.

Chronic metabolic acidosis was achieved by keeping animals on a low-alkali but high-energy diet consisting of peanuts, cornflakes, and carob tree fruit skin (PCC) for 5 days (20) and by adding 1% ammonium chloride (wt/vol) to the drinking water for the last 2 days. The electrolyte composition of the feed was determined by analysis of ashes (Institute for Animal Health and Food Quality, Kiel, Germany).

After experimentation, animals were killed by an overdose of anesthetic. Tissues for RNA analysis were quickly removed, snap frozen in liquid nitrogen, and stored at –80°C. Resulting means (±SEM) of NHE3 mRNA were compared with those of an untreated reference group (n = 24).

Statistical Analysis
Group mean values and SEM were calculated. After corroborating normal distribution and equality of variances, means were tested for significant differences by independent sample t tests. The limit of significance was at P ≤ 0.05. Correlation between selected variables and mean 95% confidence intervals was determined by regression analysis. Statistical analysis was in part performed by using SPSS 8.0 for Windows (SPSS, Inc., Chicago, IL).


   RESULTS
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 RESULTS
DISCUSSION
 REFERENCES
 
Nutrition and Renal Function
Table 1 shows the nutritional background and renal acid–base regulation in the rabbits before (n = 11) and after hypercapnic (n = 5) or NH4Cl treatment (n = 6). In addition, data from a larger control group (n = 24) were included for comparison. As a measure of food alkalinity, the cation–anion difference per 100 g was 86.9 ± 0.7 mEq in the standard food and 14.8 ± 1.6 mEq in the alkali-reduced PCC diet; the average energy contents amounted to 150 and 417 kcal, respectively.


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  		TABLE 1. NUTRITIONAL BACKGROUND AND RENAL FUNCTIONS IN THE STUDY GROUPS

 
Daily alkali and energy intake is based on food intake and composition of nutrients. With respect to fat, protein and carbohydrate content respiratory exchange ratios ranged from 0.91 to 0.93 in both types of feed.

Posthypercapnic group
After hypercapnia, there were no major changes compared with the control group or with the situation before treatment. This notion is especially important for nutritional alkali intake and fractional renal base reabsorption, which was also not different. Nevertheless, absolute bicarbonate reabsorption was significantly increased proportional to an increased amount of filtered HCO3– (HCO3–FIL = GFR · HCO3–a [mmol · L–1GFR]; see Tables 1 and 2).


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  		TABLE 2. EFFECTS OF PROLONGED HYPERCAPNIA AND CHRONIC METABOLIC ACIDOSIS ON ARTERIAL ACID–BASE CONDITIONS AND VENTILATION

 
NH4Cl group
Upon voluntary water intake, daily NH4Cl ingestion was 13.6 ± 1.7 mEq. Prolonged application of ammonium chloride in conjunction with the PCC diet converted the normal nutritional surplus of alkali into an acid load. Although the animals reduced the quantity of daily food intake, energy intake and body weight remained unchanged. Due to nutritional acidification, urinary pH decreased to unusual low values for rabbits. The GFR estimated from endogenous creatinine clearance showed a tendency toward reduction (P = 0.07), whereby the absolute reabsorption of bicarbonate was unchanged. The fractional HCO3– reabsorption amounted to nearly 100%. As in other species, the increased fractional base reabsorption during metabolic acidosis was paralleled by a rise in NHE3 mRNA as measured in renal cortices of the NH4Cl and the control group (Table 1).

Systemic Acid–Base Status and Ventilation
Mean data describing the respiration-related functions of the experimental groups are shown in Table 2. In the posthypercapnic group, the arterial pH was slightly increased, whereas systemic BE was considerably higher than under control conditions. Concomitantly, there was an increase in PaCO2, but no significant change in VA. Metabolic VCO2 did not show any difference.

Chronic metabolic acidosis observed in the NH4Cl group was characterized by distinctly lowered values of arterial pH and BE. Concomitantly, there was a small decrease in PaCO2 and considerable depression of VA by about 25%. Levels of PaO2 and lactate were not different from controls. VCO2 was significantly decreased, indicating a reduced metabolic rate.

NHE3 Expression and Ventilation
NHE3 mRNA expression was quantified in the posthypercapnic, the NH4Cl-treated, and the control groups (Table 2 and Figure 1). Subsequent to chronic hypercapnia, brainstem NHE3 mRNA expression amounted to 1.48 ± 0.55 fg cDNA/µg RNA, which was not different from the average control level. Likewise, these animals did not differ from controls with respect to VA. In contrast, the level of brainstem NHE3 mRNA was increased 2.5-fold during chronic metabolic acidosis, and VA was significantly decreased in these animals.


Figure 1
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  		Figure 1. Brainstem sodium/proton exchanger (NHE) 3 expression and ventilation during prolonged acid–base imbalance. Bars represent mean values ± SEM of untreated controls (black; n = 24) of animals after exposure to CO2-enriched air for 72 hours (dark gray; n = 5) and of those supplemented daily with ~14 mEq · ammonium chloride for 2 days (light gray; n = 6). Significance of differences compared with controls is given at levels of *P < 0.05 and **P < 0.01. Note distinctly enhanced NHE3 mRNA expression upon chronic metabolic acidosis, but not after prolonged hypercapnia.

 
As previously shown for a smaller number of measurements (9), there was an inverse correlation between VA and NHE3 expression under control conditions (i.e., normal BE and VCO2). Thus, the new data strongly corroborate this correlation (Figure 2). To our surprise, and despite the experimentally induced changes in acid–base equilibrium (Figure 1, Table 2), the mean values from both the posthypercapnic and NH4Cl-treated subgroups matched with the 95% confidence interval of this regression line. Results suggest that changes in ventilation during chronic acid–base imbalance do not monocausally depend on acid–base conditions (see below).


Figure 2
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  		Figure 2. Medullary brainstem NHE3 expression and ventilation. Alveolar ventilation (VA) as a function of NHE3 mRNA was estimated by regression analysis from repeated measurements in 24 rabbits under control conditions, including the data from Reference 9. Means (±SEM) for the posthypercapnic group (n = 5) and for the NH4Cl group (n = 6) are added. These groups appear to be matched with the 95% mean confidential interval despite experimentally induced acid–base imbalance.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study shows for the first time that brainstem NHE3 mRNA expression in rabbits is significantly up-regulated during chronic metabolic acidosis induced by ammonium chloride, whereas there is no change after prolonged hypercapnia. Although the single steps of the transduction cascade are still unknown, there is convincing evidence from studies in vitro and in vivo that the pHi of chemosensitive neurons is the adequate signal to elicit respiratory responses (1, 2, 6, 8, 22). As discussed in detail by Putnam and colleagues (3), several pH-sensitive potassium channels may link up membrane potential to pHi, and may thus serve as H+ sensors, the most sensitive of which are located in the parapyramidal region at the ventrolateral surface (23), where they can be excited with NHE3 inhibitors (24). Thus, a causal link is likely between the expression of NHE3 and respiratory control, brought about by NHE3-expressing chemosensitive neurons of the ventrolateral medulla oblongata.

Systemic Acid–Base Balance and Ventilation
Before we discuss mechanisms underlying long-term effects of hypercapnia and NH4Cl treatment, possible influences of hyperoxia on ventilation will be considered.

We are aware that central chemosensitive neurons in vitro are stimulated by oxidative stress, likely due to reduced NHE activity and impaired pHi regulation (25). During the measurements, we briefly applied oxygen-enriched air to the animals to reduce the respiratory drive by peripheral chemoreceptor activity, which, in rabbit single chemoreceptor fibers under normoxic conditions, amounted to as much as 30% of maximum hypoxic discharge (26). In line with this, it can be calculated for conscious rabbits (20) that VA decreases by approximately 12% (821 ± 34.7 to 719 ± 24.7 ml · min–1; P < 0.02) when the PaO2 is enhanced from normoxic to hyperoxic values (120–250 mm Hg). This demonstrates that central effects of hyperoxia are of minor significance for conscious rabbits, and, apart from that, cannot contribute to differences observed between the experimental groups, as oxygen-enriched air was equally applied to all animals.

Effects of Prolonged Hypercapnia
Chronic hypercapnia increased VE as long as it was applied. Under posthypercapnic reestablished eucapnic conditions, VA in our rabbits was nearly unchanged from prehypercapnic values, despite elevated PaCO2 and BE (Table 2). This compensatory metabolic alkalosis after prolonged hypercapnia appears to be a common phenomenon in several species including humans, whereby no ventilatory depression was found (for review, see Reference 27). In dogs after prolonged hypercapnia despite elevated bicarbonate buffers, ventilation was neither related to blood nor to cerebrospinal fluid, pH (CSF) (28). Rats subjected to severe hypercapnia for several months showed unchanged ventilation equal to that of the prehypercapnic levels, whenever intermittently tested in room air (21). Also, in this case there was a metabolic alkalosis during the testing periods and a concomitant rise in PaCO2 comparable to that what we found. Possibly this constellation reflects an adaptation of the metabolic CO2 production which may influence ventilation as well (see below).

Effects of Prolonged NH4Cl Ingestion
Two days of ammonium chloride treatment in conjunction with a nonalkalogenic feed induced a pronounced metabolic acidosis in conscious rabbits, which could not be prevented by even complete renal reabsorption of filtered bicarbonate (Table 1). As indicated by only slightly reduced PaCO2, this acidosis was hardly compensated by altered respiration (Table 2). Most striking, VA of NH4Cl-treated rabbits was rather decreased than increased (Figures 1 and 3) as one might expect due to the overall systemic acidosis.


Figure 3
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  		Figure 3. Vector model for respiratory baseline setting after prolonged hypercapnia and during chronic metabolic acidosis. Three "metabolic hyperbolae" (dashed lines) represent levels of elevated, normal, and diminished VCO2. (A) In the posthypercapnic group (n = 5), two factors affect VA to about the same extent in opposite directions; namely, slightly enhanced VCO2 (vertical arrow) and decreased systemic H+ (thin upper arrow). The result is nearly unchanged ventilation at higher PaCO2 (thick solid arrow). (B) In the NH4Cl group (n = 6), three factors affect VA to different degrees and in different directions: up-regulated NHE3 in the brainstem (upper thin arrow), lowered VCO2 (vertical arrow), and increased systemic H+ (lower thin arrow). The result is lowered ventilation at slightly reduced PaCO2 (thick solid arrow).

 
As suspected above, an influence on VA may also emanate from the metabolic rate. In the NH4Cl group, we found a reduction in VCO2 (Table 2). A reduced daily food intake in our rabbits during ingestion of ammonium chloride cannot account for the lowered metabolic rate, because the high energy content of the diet prevented any deficit in alimentary energy uptake (Table 1). In line with this, fasting human volunteers showed significantly reduced baseline energy metabolism by up to 22% when taking ammonium chloride for 24 hours (29). Differences in metabolic rate that we found can also not be attributed to different feed components that could reduce VCO2 at the expense of fat metabolism, as respiratory exchange ratios estimated for the two herbivore diets used in our study were equally above 0.90. As has been shown for man, levels of thyroxin are decreased (–30%) during fasting and also upon NH4Cl-induced metabolic acidosis (30), which likely lowers baseline metabolism. Likewise, acidotic inhibition of pH-sensitive enzymes, such as the phosphofructokinase regulating the glycolytic pathway (31) or pH-dependent changes of glucose utilization and glutamine formation, may be plausible explanations (32, 33).

The lack of augmented ventilatory drive by chronic metabolic acidosis that we found in conscious rabbits is in contrast to reports on humans that outlined increases (albeit small) in ventilation upon exposure to NH4Cl (34, 35). There was general agreement that the pH of cerebrospinal fluid remains remarkably stable under conditions of long-term acid load achieved by either NH4Cl (27) or hydrochloric acid (36). In the latter study, conscious dogs did not significantly hyperventilate after 1 week of dietary acid load, as long as secondary hypocapnia was allowed to occur. On the other hand, the loss of bicarbonate during prolonged ingestion of hydrochloric acid was considerably reduced when secondary hypocapnia was prevented by inhaled CO2 (36, 37). This raised the idea that hyperventilation in response to chronic metabolic acidosis could be maladaptive as it would waste bicarbonate buffer. Our data also suggest that a mechanism that limits ventilation, and thus keeps PaCO2 at a nearly normal level, may be beneficial under these conditions. Alterations of central chemosensitivity due to changes of key molecules, such as NHE3, may provide at least one possible explanation.

Brainstem NHE3 mRNA Expression
Effects of prolonged hypercapnia
Brainstem NHE3 mRNA expression was found to be unchanged after prolonged hypercapnia (Figure 1). As previously discussed here, enduring hypercapnia for 3 days was briefly interrupted to obtain eucapnic respiratory baseline values and was reestablished before tissue sampling. Because the half-life of NHE3 mRNA amounts to several hours (38), our finding of no NHE3 mRNA up-regulation upon chronic respiratory acidosis cannot be attributed to possible mRNA instability during short-term interruption of hypercapnia.

There are also no reports on altered NHE3 abundance in the kidney during prolonged respiratory acid–base changes (15–17). These findings could explain why the fractional (but not the absolute) renal base reabsorption (Table 1) remained the same before and after chronic hypercapnia in our experiments. Although a change in brainstem NHE3 abundance can be excluded as a possible factor to adjust ventilation under these conditions, no direct conclusions can be drawn concerning exchange rates and pHi regulation in central chemosensitive sites.

It is possible that higher maximal exchange velocities are elicited by chronic respiratory acidosis, as has been observed in kidney tubules (39). However, if this was in fact the case for central chemosensors, ventilation should have been more visibly decreased below the reference range in Figure 2.

Effects of prolonged NH4Cl ingestion
Upon chronic metabolic acidosis, the mean level of brainstem NHE3 mRNA increased 2.5-fold (Table 2; Figure 1). In kidney cortices of this animal collective, we found a parallel increase in NHE3 expression (Table 1), thus confirming previous findings (13, 14). Although we have no direct evidence that the level of NHE3 mRNA in the brainstem reflects NHE3 protein abundance, this is rather likely from studies on other NHE3-expressing tissues (e.g., renal epithelium cells), where elevated NHE3 mRNA expression upon chronic metabolic acidosis approximately paralleled protein expression (40–42).

It has been suggested that increases in NHE3 abundance upon chronic metabolic NH4Cl acidosis rely on an acid sensor, or may be elicited more directly by high levels of renal NH4+ (43). However, pH regulation in brain and CSF is partly uncoupled from systemic acid–base balance (see above), such that neurons may be largely prevented from perceiving stimulatory pH changes during chronic metabolic acidosis. In addition, the finding that enhanced NHE3 expression did not occur upon respiratory acidosis argues against a cellular acid sensor. Further studies are needed to clarify why NHE3 gene expression appears to be rather equivalently regulated in brainstem and kidney.

Possible Mechanisms Underlying NHE3 Up-regulation upon Chronic Metabolic Acidosis
At present, we can only speculate on the mechanism underlying NHE3 up-regulation in the brainstem. A control of the NHE3 promoter is provided by cis-acting elements binding to upstream regions. Aside from transcription factors like SP1, SP3, AP-2, or PMA (44, 45), hormones were shown to initiate or enhance NHE3 mRNA transcription in vitro, at least in kidney cell cultures (46). Several nonsteroidal and steroidal hormones, such as thyroxin, glucocorticoids, insulin, and angiotensin II, can promote activity and expression of renal NHE3 (38, 40–42, 47). Whereas plasma levels of insulin and angiotensin II invariably rise during experimentally induced chronic NH4Cl– acidosis (49, 50), thyroxin levels are considerably reduced (30), and the responses of corticosteroids are equivocal (48–50). Therefore, one possible candidate for a systemic control of NHE3 in the brainstem is angiotensin II, as the brain renin–angiotensin system, as well as neurons in the area postrema, were shown to be influenced by circulating systemic angiotensin II (51). However, at present, it remains open whether the up-regulation of NHE3 mRNA that we found in kidney and brainstem is a direct effect on NHE3-producing cells (e.g., caused by the ammonium load, acidosis, or changed metabolic pathways) and/or an indirect effect that involves systemic hormonal responses.

A Multifactorial Model of Respiratory Baseline Setting
With respect to the inverse correlation between brainstem NHE expression and VA, it is important to note that this relationship has been established for constant levels of metabolic rate and systemic acid–base balance (9). Although the mean value for chronic metabolic acidosis seemingly matches the 95% confidence interval of the regression line under control conditions (Figure 2), additional factors must be considered.

Figure 3 shows a vector model in which the sum vector determines the final set point of breathing control during chronic metabolic acid–base disturbances. Aside from acid–base changes, brainstem NHE3 expression and metabolic rate were incorporated by single vectors. In the case of chronic metabolic acidosis (Figure 3B), a ventilatory depression of about 21% due to elevated brainstem NHE3 was estimated from the relationship between baseline VA and NHE3 shown in Figure 2. Further reduction of VA (by ~22%) was derived from the decrease in metabolic rate (Table 2). Finally, a ventilatory drive by elevated H+ of about 18% can be discerned, likely elicited to a major degree by peripheral chemoreceptors (52), whose stimulatory contribution to ventilation was blunted by the former factors. Because up-regulation of NHE3 in central chemosensitive neurons would extrude H+ and limit their firing rate, ventilation seems to be shifted into opposite directions by low systemic and high central pHi. Thus, overexpression of brainstem NHE3 may finally limit "maladaptive" hyperventilation as outlined above. On the other hand, given no change in NHE3 expression but a small tendency to enhance CO2 production after prolonged hypercapnia, the unmasked ventilatory depression by compensatory metabolic alkalosis would come to about –14% (Figure 3A).

Taken together, the present data strengthen the idea that NHE3 acts as a functional pHi-regulating membrane transporter in CO2/H+-sensitive brainstem neurons. Its level of expression in the brainstem appears of functional significance for central respiratory control under normal and disturbed acid–base conditions. As a part of the central controller, NHE3 overexpression induced by chronic metabolic acidosis may limit reactive hyperventilation, thus preventing hypocapnia and loss of bicarbonate buffer.


   Acknowledgments
 
The clinical laboratory facilities provided by P.D. Dr. Hermann Kalhoff and the skillful technical assistance of Patricia Freitag are gratefully acknowledged.


   FOOTNOTES
 
Originally Published in Press as DOI: 10.1164/rccm.200703-347OC on June 28, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form March 1, 2007; accepted in final form June 26, 2007


   REFERENCES
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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