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1Section of Respiratory Medicine, Department of Pediatrics, and 4Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520; 3Molecular Medicine and Renal Units, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215; and2Section of Respiratory Medicine, Department of Pediatrics, and5Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York 10461
Submitted 27 November 2002 ; accepted in final form 27 March 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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brain; cyclical hypoxia; proteins; central nervous system
Ionic and pH homeostasis in the CNS is controlled in major part by two families of acid-base transport proteins (5, 12, 16, 47). The Na/H exchanger (NHE) family presently consists of at least eight members (NHE1 to NHE8) (15, 21, 54). The bicarbonate (HCO3)-dependent, acid-base transport protein family includes the Na+-independent chloride (Cl)/ HCO3 or anion exchangers (AE1–3 and perhaps also the more distantly related AE4) (2, 31, 32), the Na-dependent Cl/HCO3 exchanger (NDCBE) (5), and a rapidly expanding number of Na-HCO3 cotransporters (NBC) (10, 52). Within the CNS, the ubiquitously expressed NHE1, NDCBE, AE3, and NBC are believed to be major contributors to acid-base balance in neurons and glia (5, 12, 16, 34, 47, 56, 57). NHE1, NDCBE, and some NBCs are presumed to function as acid extruders, whereas AE3 and some NBCs, depending on their stoichiometry, are acid loaders (32, 52). There is considerable evidence that acute and chronic hypoxia and ischemia produce decreases in neuronal and glia intracellular pH (pHi) (30, 33, 40). Additionally, intermittent reductions in fetal blood flow induce decreases in fetal tissue and blood pH and persistent elevated lactic acid levels (19, 45). Furthermore, Nagata et al. (39) have reported that brain lactate is slightly elevated and blood pH moderately decreased during intermittent hypoxic-ischemic insult in the neonatal rat model. Because acute and chronic hypoxia are known to induce changes in ionic homeostasis (Na+, Ca2+, H+) (6, 26, 47) and especially that of pHi and extracellular pH (30, 40), we hypothesized that CIH would also cause changes in acid-base protein expression and their regulation within the CNS. We have, therefore, performed studies in mice to examine the effect of CIH on CNS pH regulatory protein expression and, in particular, on acid-base transporters using Western blotting.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Pregnant CD-1 mice were obtained from Charles River (Wilmington, MA). Extreme care was exercised in the handling of these animals, and the minimal number of animals that was absolutely required was used in this study. This study was conducted in conformity with the Guiding Principles for Research Involving Animals and Human Beings and was approved by the Yale Animal Care and Use Committee.
CIH
The paradigm utilized to generate mice that were exposed to CIH has been previously described (25) and mimics what is seen in severe OSAHS in children and adults (3, 48). In brief, three to four groups of eight postnatal day (P)02–P03 mice of both sexes were placed into an environmental chamber with their dam for each 28-day CIH period. Food and water were provided ad libitum within the chamber, and temperature was maintained relatively constant at 
22°C.
To mimic the episodic nature of diseases such as periodic sleep apnea and OSAHS (3, 37), mice were exposed to alternating cycles of 2 min of 6.0–7.5% O2 followed by 3 min of normoxia, which were generated by balances of 100% N2
and 100% O2 gases. CO2 levels and humidity were maintained near physiological levels by the use of Drierite and a port that permitted equilibration of pressure within the chamber. This cycle was maintained for 8 h during the day, for a period of 28 days (4 wk). Control mice, with their dams, were maintained in the same environment as the hypoxia-exposed animals, but breathed room air (21% O2) of equivalent humidity for the duration of the exposure period.
Materials
NHE1 protein expression was determined by Western blotting utilizing a mouse monoclonal antibody to NHE1 (anti-NHE1; MAb 4E9) derived from a fusion protein containing the amino acids 514–818 of porcine NHE1 (8). The guinea pig/opossum polyclonal antibody to NHE3 was generated against the carboxy terminal 40 amino acids (aa 793–832) of rabbit kidney NHE3 (7), whereas the NHE4 protein was detected with a mouse monoclonal antibody (anti-NHE4; MAb 11H11) directed against a portion (amino acids 565–575) of the rat NHE4 (43).
The NBC family of proteins has been categorized as NBC1–4. NBC1 is an electrogenic cotransporter that is transcribed as at least three variant mRNAs encoding as many polypeptides. kNBC1 was first cloned from kidney (46, 52) and was later found in brain, eye, and many other tissues. pNBC1 was first cloned from pancreas (1) and was later found in heart (13), eye, and other tissues. kNBC1 and pNBC1 differ only in their amino-terminal sequences. bNBC1 was first cloned from brain (5a). bNBC1 and pNBC1 differ only in their carboxy-terminal sequences. We have utilized, in our study, three rabbit polyclonal anti-NBC antibodies that detect all reported variants of NBC1 (10, 52). 1) Anti B1B-NBC recognizes the unique carboxy-terminal 61 amino acids of the bNBC1 and so does not recognize either kNBC1 or pNBC1 (5a). 2) Anti K1A-NBC recognizes the carboxy-terminal 46 residues shared by kNBC1 and pNBC1 and so does not recognize bNBC1 (5a). 3) Anti RK-NBC5 was raised against the carboxy-terminal 108 residues shared by kNBC1 and pNBC1 (50, 52). The first 62 of these 108 residues are also shared with bNBC1. Thus anti-RK-NBC5 can, in principle, recognize all three variant NBC1 polypeptides. The affinity-purified rabbit polyclonal antibody to the carboxy-terminal 12 residues of AE3 detects a protein of 180 kDa and does not cross-react with the abundant AE2 of choroid plexus (55). The antibodies utilized in these studies were kindly provided by Dr. Dan Biemesderfer (anti-NHE1, NHE3, and NHE4; Yale University School of Medicine), Dr. Walter Boron (anti-RK-NBC, K1A-NBC, and B1B-NBC; Yale University School of Medicine), and Dr. Seth Alper (anti-AE3; Harvard Medical School, Boston, MA).
Data expressed as the ratio of acid-base transporter polypeptide density to actin abundance are reported as means ± SE. Within any given brain region, actin abundance per milligram total membrane protein did not change with CIH. Differences between control and intermittently hypoxic groups were assessed via the Student's t-test and the Wilcoxon rank sum test. Differences between means were considered statistically significant if P < 0.05.
Immunoblotting
Tissue preparation. Mice were deeply anesthetized with halothane (Halo Carbon, Halocarbon Laboratories, Melville, NY) via inhalation (a few drops of halothane in a closed chamber), weighed, and quickly decapitated with a guillotine. The brains were rapidly removed from the cranium and placed in an ice-cold lysis buffer (in mM: 200 mannitol, 80 HEPES, and 41 KOH), pH 7.5. The protease inhibitors pepstatin A (1 µM), leupeptin (1 µM), PMSF (230 µM), and EDTA (1 mM) (Sigma, St. Louis, MO) were added to the lysis buffer just before usage. The brains were then segregated into four components, i.e., the cerebral cortex (CX), the hippocampus (HC), the cerebellum (CB), and the brain stem-diencephalon (BD). The CX includes the superficial lobes (frontal, parietal, occipital, and temporal) of the cerebrum minus the HC. The BD represents subcortical structures, such as the basal ganglia, thalamus, and hypothalamus, the brain stem proper, and part of the cervical spinal cord. For each lane in an immunoblot, we pooled these various regions from three to four animals. In Figs. 2, 3, 4, 5, 6, 7, 8, the n values represent the number of immunoblot lanes examined for each antibody and brain region, each of which represents tissues pooled from three to four animals. Pooled regions of the CNS were briefly removed from the lysis buffer to determine the wet tissue weight and transferred to 4x volume/weight of lysis buffer for the homogenization step of the microsomal preparation.
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Membrane preparation. Crude microsomes were prepared from each of the four CNS regions, according to the method of Grassl and Aronson (24). Tissues were homogenized by 10–20 strokes with a Teflon-glass homogenizer operating at 2,000 rpm (Thomas Scientific, Swedesboro, NJ). The homogenate was then centrifuged at 1,000 g for 10 min to remove cellular debris. The supernatant was withdrawn and centrifuged at 100,000 g in a Beckman SW40T rotor for 1 h. The resulting pellet was resuspended in 200–1,000 µl of lysis buffer and stored at -80°C until used.
SDS-PAGE and Western blotting. Protein concentrations of pooled membranes were assayed by using a DC protein assay kit (Bio-Rad, Hercules, CA). Membrane fractions (30 µg) of each region were then solubilized in sample loading buffer (2% SDS, 20% glycerol, 100 mM 
-mercaptoethanol, 5 mM Tris, pH 6.8) and separated by SDS-PAGE utilizing 7.5% polyacrylamide gels. Proteins were then transferred to polyvinylidene fluoride (Immobilin-P, Millipore, Bedford, MA) membranes at 300 mA for 4–6 h in a Trans-Blot cell (Bio-Rad).
Membranes were incubated in 5% nonfat dry milk (Carnation, Nestle Food, Glendale, CA) in PBS (in mM: 148.9 NaCl, 2.8 NaH2PO4, 7.2 Na2HPO4, pH 7.4) with 0.1% Tween 20 (American Bioanalytical, Natick, MA) for 1–2 h to block nonspecific proteins. The membranes were then incubated overnight at 4°C in 5% nonfat dry milk/PBS containing one of the following primary antibodies: mouse monoclonal anti-NHE1 hybridoma supernatant (dilution factor of 1:4), affinity-purified guinea pig/opossum monoclonal anti-NHE3 IgG (1:1,000), mouse monoclonal anti-NHE4 hybridoma supernatant (1:2), rabbit polyclonal anti-NBC serum (RK-NBC 1:400; K1A-NBC 1:400; B1B-NBC 1:2,000), and affinity-purified rabbit polyclonal antibody to AE3 (1:1,000). Membranes were subsequently rinsed five times for 3, 3, 15, 5, and 5 min, respectively, in 5% nonfat milk/PBS and then incubated for 1 h at room temperature in 5% nonfat dry milk/PBS containing a secondary antibody (anti-mouse, anti-rabbit, or anti-guinea pig IgG, whole molecule) linked to horseradish peroxidase at a dilution of 1:2,000 (Zymed, South San Francisco, CA). Membranes were again rinsed, utilizing the same protocol as above, and protein signal detection was achieved with the ECL chemiluminescence system (Amersham, Little Chalfont, UK). Membranes were stripped, according to the manufacturer's recommendation, and reprobed with a goat monoclonal antibody to actin (1:1,000) (Santa Cruz, CA) to act as an internal control. Scanning densitometry of immunoblot films was performed on a Personal Densitometer SI scanner (Molecular Dynamics/Amersham Biosciences, Sunnyvale, CA) and analyzed by using the ImageQuaNT image analysis software (Molecular Dynamics).
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Mice exposed to CIH demonstrated lower body weights throughout the period of hypoxic exposure (unpublished observations). Mice exposed to CIH for 28 days weighed 16.7 ± 0.7324 g at the end of the hypoxic period, whereas control mice weighed significantly more (23.05 ± 0.6399 g) after the same period of time (P < 0.001) (Fig. 1).
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The Effect of CIH on NHE
There was a robust and ubiquitous expression of NHE1 protein in the mouse CNS (Fig. 2A) as in the rat CNS (17). A high level of expression of NHE1 existed in the BD, and lesser quantities of expression in the CX, HC, and CB. Intermittent hypoxia of 28-day duration elicited a decrease in NHE1 protein expression in three of the four brain regions examined (Fig. 2B), with one of them being significant in the CB (23%). Also, similar to rat CNS (17) are low levels of expression of NHE4 (Fig. 3A) and NHE2 proteins (not shown) throughout the neuraxis. However, there was little or no change in the expression of NHE2 and NHE4 (Fig. 3B) between control mice and others that were subjected to CIH. There was little or no expression of the NHE3 isoform in the CX, HC, or BD of the mouse, as in the rat (unpublished observations from our laboratory) (Fig. 4A). However, there was a robust expression of the NHE3 protein in the CB (Fig. 4A), which corroborates the mRNA in situ hybridization and Northern blot data reported by Ma and Haddad (36). Similar to the response of the NHE1 protein to 28 days of intermittent hypoxia, NHE3 protein expression was reduced in the HC (44.5%) and BD (16.9%) (Fig. 4B). In the CB, the decrease in NHE3 expression (33%) was significant (Fig. 4B).
The Effect of CIH on HCO3-dependent Acid-Base Transporters
AE3 is a Cl/HCO3 exchanger highly expressed in brain parenchyma (32). In control mice, there was a substantial expression of AE3 protein in the CX, HC, and BD but less in the CB (Fig. 5A). CIH induced some degree of decrease in the abundance of AE3 polypeptide in all brain regions studied (Fig. 5B). Whereas the decrease in AE3 was slight in the BD (12%), more substantial decreases occurred in the CX (27%), HC (20%), and CB (30%). However, only the decrease in AE3 protein expression in the HC was statistically significant.
In general, the response of NBCs to CIH resembled that of NHE1 and NHE4 in that there was a trend toward a decrease in RK-NBC protein expression in all regions of the CNS (Fig. 6A). There was a significant decrease, however, in the expression of RK-NBC-reactive protein in the CB (33%) (Fig. 6B). The K1A-NBC antibody (Fig. 7A) was designed to detect proteins that are also recognized by the RK-NBC antibody but not by the bNBC-specific B1B-NBC antibody (5a). NBC polypeptides detected by both K1A-NBC (Fig. 7B) and RK-NBC antibodies (Fig. 6A) were most abundant (as normalized to actin) in the CB. CIH significantly decreased the abundance of K1A-NBC-reactive protein only in the HC (22%) (Fig. 7B). In contrast to the decreased expression of protein detected by RK-NBC and K1A-NBC antibodies, NBC protein levels detected by the brain-specific B1B-NBC were either not changed or increased in response to 28 days of intermittent hypoxia (Fig. 8A). This increase in B1B-NBC-detected proteins was substantial in the CX (30%) but less marked in the CB (14%) and the BD (18%) (Fig. 8B).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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In the present study, the paradigm of cyclical or episodic hypoxia was selected so as to most closely emulate that seen in the clinical condition of OSAHS (3, 37, 48). As a result of 28 days of CIH, we observed, in at least one brain region, an overall decrease in the expression of a majority of the acid-base transport proteins examined in the mouse CNS. It is especially noteworthy that the CB appears to be the most susceptible CNS region to CIH-induced protein downregulation. For example, the acid extruders NHE1 and NHE3 (5, 15) showed a trend toward a decrease in all CNS regions and decreased significantly in the CB. This decrease in expression was not nonspecific, as the NHE isoforms 2 and 4 (NHE2 and NHE4), which demonstrate parallel developmental and regional distribution profiles in the rat (17) and mouse, do not demonstrate any changes in expression in response to CIH in the mouse CNS. Furthermore, our observation that these acid-base transporters are altered in the CB and HC and not in other regions is important to note, because these areas are known to be metabolically very active and vulnerable to O2 deprivation (11, 26).
CIH also produces general decreases in the expression of Na+-HCO3-acid-base transporters (NBC proteins). Although the role of NBC proteins as acid-extruding or acid-loading transporters is determined by several variables (e.g., the stoichiometry, the membrane potential, and ionic activities), the expression level is by itself an important determinant of the activity of the transporter. NBCs, as detected in our studies by antibodies directed against an electrogenic NBC (NBC1) (5a, 50), and the AE3 were significantly decreased only in select CNS regions. The RK-NBC antibody detects a significant decrease in NBC protein expression in the CB in response to CIH. The K1A-NBC antibody detects a significant decrease in NBC protein expression in the HC. However, NBCs detected by the brain- and neuron-specific antibody B1B-NBC (5a) were increased or unchanged. These data suggest that differential expression of NBC isoforms occurs in discrete CNS regions and that these region-specific isoforms are variably regulated by hypoxic exposure. We believe that this is the first report that addresses the effect of CIH on acid-base transport protein expression in the CNS.
Hypoxia similarly induces downregulation of other membrane proteins, such as Na-K-ATPase, the epithelial Na channel, voltage-gated Na+ channels, and N-methyl-D-aspartate receptors within the CNS (14). Hypoxia-ischemia and excitotoxicity have been reported to decrease protein synthesis, especially in the HC (44). However, unlike viral infections that induce a general decrease in all protein synthesis, and unlike the inhibition of protein synthesis that occurs during combined O2-glucose deprivation in the HC, CIH has a variegated effect on acid-base protein expression in the mouse CNS. This regional variation further suggests that the decrease in expression of selected acid-base transporter proteins associated with CIH in this study is not nonspecific.
If CIH lowers the acid-extruding capacity of neurons, CIH may then render CNS cells even more acidic than would occur if hypoxia-responsive metabolic adjustments alone were at play. It has been reported that, during hypoxia-ischemia and reperfusion, mild-to-moderate acidosis may contribute to neuronal survival (20), whereas severe acidosis acts synergistically with hypoxia-ischemia to induce neuronal injury and cell death (30, 40, 51). It has also been reported that blockade of NHE or NDCBE function leads to intracellular acidification of cortical neurons (5, 9, 34). Therefore, it is conceivable that intracellular acidification during CIH in conjunction with a decrease in available acid-extruding proteins (NHE1, NHE3, NBC) could render neurons more acidotic and, therefore, more susceptible to injury. For example, compromised acid extrusion has the capacity to impair neuronal function via H+-mediated influences on electrotonic coupling, neurotransmitter release, second-messenger systems, and channel activity (9). However, there is still a controversy as to the effect of CIH on pHi and the resulting effects. On the one hand, there have been several reports indicating that CIH induces neuronal injury and apoptosis (4, 38). For example, in a paradigm that is equivalent to the one used in this study, Gozal et al. (23) reported that CIH induced apoptosis in the hippocampal CA1 but not the CA3 region and marked cellular dystrophy and cognitive impairment that persisted for 2 wk beyond the hypoxic exposure. On the other hand, Nagata et al. (39) have reported that, even though the acidemia and lactate production induced by CIH are relatively minor compared with the effect of continuous hypoxia-ischemia, brain damage caused by repetitive hypoxia-ischemia is more pronounced than that caused by an equivalent period of continuous hypoxia-ischemia. It is, therefore, evident that the effect of CIH on pHi needs to be determined to more exactly ascertain the effect of the decrease in acid-base transporter expression on hypoxia-induced injury. Additionally, functional studies of exchanger and transporter activity under conditions of CIH need to be performed to understand more completely the effect of CIH on pHi and acid-base homeostasis.
The decrease in acid-base protein expression resulting from CIH may be part of a more "global" cellular strategy during chronic hypoxic stress, indicative of a more generalized decrease in cellular protein synthesis, which would serve to minimize the expenditure of metabolic energy reserves already compromised by hypoxic exposure. Hochachka (29) and other authors have postulated that a decrease in protein anabolism may serve a protective role during hypoxia (28), whereas Ma and Haddad (35) have suggested that there is a hierarchy of proteins whose expression is either maintained or diminished during hypoxia, depending on the needs of the cell. The present data on acid-base transporter proteins are consistent with the latter hypothesis.
In summary, we have shown that several pH regulatory ion transport proteins of the mouse brain exhibited decreased abundance in specific brain regions after in vivo exposure to CIH. This response could participate in a cascade of events that lead to cellular injury. Alternatively, this decrease in protein expression may be reflective of an adaptive cellular response to chronic hypoxia.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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This work was supported by National Institutes of Health Grants HL-07778, HD-32573, and NS-35918 and the Parker B. Francis Fellowship.
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FOOTNOTES |
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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. Section 1734 solely to indicate this fact.
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