1 Center for Oral Biology in the Aab Institute of Biomedical Sciences and Departments of 3 Dentistry and 2 Neurobiology and Anatomy, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Little is known of the functional properties of the mammalian, brain-specific Na+/H+ exchanger isoform 5 (NHE5). Rat NHE5 was stably expressed in NHE-deficient PS120 cells, and its activity was characterized using the fluorescent pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. NHE5 was insensitive to ethylisopropyl amiloride. The transport kinetics displayed a simple Michaelis-Menten relationship for extracellular Na+ (apparent KNa = 27 ± 5 mM) and a Hill coefficient near 3 for the intracellular proton concentration with a half-maximal activity at an intracellular pH of 6.93 ± 0.03. NHE5 activity was inhibited by acute exposure to 8-bromo-cAMP or forskolin (which increases intracellular cAMP by activating adenylate cyclase). The kinase inhibitor H-89 reversed this inhibition, suggesting that regulation by cAMP involves a protein kinase A (PKA)-dependent process. In contrast, 8-bromo-cGMP did not have a significant effect on activity. The protein kinase C (PKC) activator phorbol 12-myristrate 13-acetate inhibited NHE5, and the PKC antagonist chelerythrine chloride blunted this effect. Activity was also inhibited by hyperosmotic-induced cell shrinkage but was unaffected by a hyposmotic challenge. These results demonstrate that rat brain NHE5 is downregulated by activation of PKA and PKC and by cell shrinkage, important regulators of neuronal cell function.
pH regulation; amiloride; sodium-proton exchange; sodium/hydrogen
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
SODIUM/HYDROGEN EXCHANGER isoform 5 (NHE5) is a member of the mammalian family of integral membrane proteins that are involved in the regulation of intracellular pH (pHi), cell volume, and electrolyte transport (reviewed in Refs. 14, 30, 44, and 52). Other known members of this family include four additional plasma membrane isoforms (NHE1-NHE4; see Refs. 32, 36, and 45) and a mitochondrial isoform NHE6 (28). The deduced amino acid sequence and predicted membrane organization of NHE5 is similar to other members of the Na+/H+ exchanger gene family, being most like NHE3 (2, 3). However, in contrast to the epithelial NHE3, high-level expression of NHE5 mRNA is restricted exclusively to the brain (2, 20), suggesting that this isoform performs a specialized role in this tissue.
Individual NHE isoforms have unique functional properties including different exchange kinetics, pharmacological characteristics, cellular localization, and tissue expression (2, 3, 13, 30, 32, 39, 44, 45, 52). The regulation of the different NHE isoforms by second messengers is distinct as well. Two major signaling pathways often involved in regulating the activity of Na+/H+ exchangers include the serine/threonine kinases protein kinase A (PKA) and protein kinase C (PKC) (18, 23, 42, 47, 53). The unique sensitivities of the different isoforms to phosphorylation reside in regulatory domains located within the cytoplasmic carboxy terminus, the region where Na+/H+ exchangers differ most significantly in their primary sequences. Deletion analysis and domain swapping experiments have confirmed that the carboxy terminus contains the elements sensitive to second messenger regulation (12, 24, 51).
Most tissues express multiple NHE isoforms, and the brain is no exception (2, 20, 32, 45). This makes it difficult, if not impossible, to functionally isolate and characterize NHE5 in native cells. Therefore, the present study investigates the functional properties of rat NHE5 by stable expression of its cDNA in PS120 cells lacking Na+/H+ exchanger activity. NHE5 activity was compared with the Na+/H+ exchanger activity in PS120 cells expressing NHE1 or NHE3. The functional properties and ethylisopropyl amiloride (EIPA) sensitivity of NHE5 were qualitatively most like NHE3. Furthermore, the inhibition of NHE5 activity in response to cell shrinkage, PKA, and PKC stimulation suggests that the regulation of this brain-specific Na+/H+ exchanger may be critical to the activity of neurons in the central nervous system.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials.
2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein
(BCECF)-AM, carboxyseminaphthorhodafluor (SNARF)-1-AM, and nigericin
were from Molecular Probes (Eugene, OR). Dulbecco's modified Eagle's medium (DMEM) and G418 were from GIBCO BRL (Grand Island, NY). Phorbol
12-myristate 13-acetate (PMA), 4-PMA, forskolin,
1,9-dideoxyforskolin (1,9-DDF), 8-bromo-cAMP (8-Br-cAMP),
N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), and chelerythrine chloride were purchased from Biomol Research
Laboratories (Plymouth Meeting, PA). All other reagents were purchased
from Sigma (St. Louis, MO). Stock solutions of forskolin, 1,9-DDF, PMA,
4
-PMA, chelerythrine chloride, H-89, and EIPA were prepared in DMSO.
8-Br-cAMP and 8-Br-cGMP were dissolved in deionized, distilled
H2O.
Transfection and stable expression of rat NHE5. The PS120 cells, which lack endogenous Na+/H+ exchange activity, were maintained in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin (50 U/ml)/streptomycin (50 µg/ml). Rat NHE1, NHE3, and NHE5 cDNAs were inserted into the pCMV/SEAP vector (Clontech, Palo Alto, CA), and stable expression was established in NHE-deficient PS120 cells as previously described (2). Cells were selected for stable expression with G418 (1,000 U/ml) and further selected for expression of Na+/H+ exchanger activity by the "H+ killing" method (43). Transfected cells were maintained in medium containing G418, and the H+ killing procedure was repeated every 4-5 days to eliminate revertants.
RNA isolation and RT-PCR of NHERF and ezrin. Total RNA was prepared from confluent 100-mm plates of culture cells or from 500 mg of hamster kidney using TRIzol reagent (Life Technologies, Rockville, MD). Total RNA (1 µg) was then reverse-transcribed with an oligo(dT) primer using Stratagene's RT-for-PCR kit (La Jolla, CA) as recommended. Five microliters of the diluted first-strand cDNA was subjected to PCR in a final volume of 25 µl that contained 10 mM Tris · HCl, pH 8.8, 50 mM KCl, 1.5 mM MgCl2, 0.08% Nonidet P-40, 200 µM of each dNTP, and 0.4 µM of one of the amplimer pairs described below. Each reaction was brought to 94°C for 1 min, then to 72°C while 0.3 µl of Taq polymerase was added, and then cycled at 94°C for 15 s, 55°C for 15 s, and 72°C for 30 s. After the appropriate number of cycles, the reactions were incubated at 72°C for 5 min. The following amplimer pairs were generated from mouse sequences: 1) Na+/H+ exchanger regulatory factor (NHERF): 5'-AGCAATGGAGAGATACAGAAGG-3' and 5'-TAAGGTGAGGGAAGAACAGG-3' and 2) ezrin: 5'-TCACACAGAAGCTCTTCTTCC-3' and 5'-AGATGTTCCTGATCTCACTCC-3'. The NHERF and ezrin reactions were cycled 35 times (hamster templates), and the reaction products were separated by size on a 2% agarose gel. cDNA (Qiagen, Chatsworth, CA) was prepared and used directly for cycle DNA sequencing with ABI BigDye terminator mix (Foster City, CA) and thermostable DNA polymerase on an MJResearch autosequencer (Watertown, MA). The reactions were run by the University of Rochester Core Nucleic Acids Facility.
pHi measurements. The pHi of individual cells plated on glass coverslips (<50% confluent) was monitored using the pH-sensitive dye BCECF on the microscope stage of an imaging platform (Axon Instruments, Foster City, CA) as previously described (27). BCECF-containing cells were acid loaded using the NH4Cl prepulse technique (35) to monitor the Na+-dependent recovery of pHi. The advantage of this approach is that physiological concentrations of extracellular Na+ are used to investigate the effects of stimulation. Briefly, coverslips were superfused with a physiological salt solution containing 60 mM NH4Cl (NaCl was replaced by NH4Cl) for 10 min and then switched to a Na+-free salt solution (except where indicated) to produce an acid load. Approximately 3 min later, extracellular Na+ was restored to initiate Na+/H+ exchanger-mediated pHi recovery. The physiological salt solution contained (in mM) 135 NaCl, 5.4 KCl, 0.4 KH2PO4, 0.33 NaH2PO4, 10 glucose, 20 HEPES, 1.2 CaCl2, and 0.8 MgSO4, and the pH was adjusted to 7.4 with Tris base. Na+ was replaced by N-methyl-D-glucamine (or choline chloride where indicated in the figure legends). In experiments to test the regulation of exchanger activity by changes in osmolarity, 55 mM NaCl was removed (hypotonic solution; zero sucrose) and replaced with either 110 mM sucrose (isotonic solution) or with 320 mM sucrose (hypertonic solution). The osmolality (mosmol/kgH2O) of all solutions was determined using a vapor pressure osmometer (Wescore 5500; Logan, UT). The pH-sensitive dye SNARF-1 was used to test the sensitivity of NHE5 activity to harmaline, a Na+/H+ exchange inhibitor that interferes with the BCECF fluorescence signal.
Calibration of the pHi signal was accomplished by the high potassium-nigericin technique (40). Recovery rates (Data presentation. All data are reported as means ± SE for the indicated number of cells (n). Each experiment was repeated three times on different days (3 different coverslips each day for a total of 9 coverslips/experiment) unless otherwise indicated. Data were analyzed by a two-tailed Student's t-test, and differences between test and control values at P < 0.05 were considered statistically significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Kinetic properties of rat NHE5.
To verify that the functional properties of rat NHE5 are similar to
human NHE5 (39) under "physiological" conditions
(i.e., in a high external Na+ concentration), we stably
expressed a cDNA containing its open reading frame in NHE-deficient
PS120 cells and characterized the resulting
Na+/H+ exchanger activity using the fluorescent
pH-sensitive dye BCECF. For comparative purposes, similar experiments
were performed with PS120 cells expressing rat NHE1 and NHE3, the
properties of which have been previously described (33,
49). pHi recovery from an acid load was not observed
in nontransfected PS120 cells (data not shown, see Ref.
2). In contrast, an intracellular
Na+-dependent pH recovery was activated in acid-loaded
cells transfected with NHE1, NHE3, or NHE5 (Fig.
1). The Na+-dependent pH
recovery observed in NHE5-expressing cells was inhibited by the
amiloride analog EIPA, but this block required relatively high
concentrations of antagonist (Fig. 1, A, and as summarized in D). Previous studies have shown that rat
Na+/H+ exchangers display distinct
sensitivities to EIPA (33, 49). Like rat NHE3 (Fig.
1B), NHE5 was substantially more resistant than NHE1 to this
inhibitor (Fig. 1C). These results verify that under the
physiological conditions of the current studies, the EIPA sensitivity
of NHE1 NHE5 > NHE3. Rat NHE1, NHE2, and NHE3 are also
blocked by harmaline, an inhibitor of Na+/H+
exchange unrelated to amiloride: K1/2 = 0.14, 0.33, and 1 mM, respectively (29, 31, 49).
Harmaline, up to 0.3 mM, failed to inhibit NHE5 activity (Fig.
1D). We used SNARF-1 for this latter experiment because this
pH-sensitive dye is resistant to fluorescence interference by harmaline
at the concentrations used.
|
|
|
|
Regulation of NHE5 by cell volume.
In many cell types, including neuronal cells (1), the
regulation of Na+/H+ exchanger activity can be
associated with changes in cell volume. Osmotic shrinkage often results
in activation of Na+/H+ exchange, which is
generally coupled with Cl/HCO
|
Regulation of rat NHE5 by PKC.
PKC activation enhances the Na+/H+ exchanger
activity of rat NHE1 and NHE2 but inhibits NHE3 (18).
Figure 6A (and as summarized in Fig. 7A) shows that
activation of PKC by the phorbol ester PMA inhibited the activity of
NHE5 >70%. In contrast, the inactive phorbol ester, 4-PMA,
produced a relatively small decrease in the pH recovery rate of NHE5
that was not statistically significant (Figs. 6B and
7A). In agreement with previous studies in which rat NHE1
and NHE3 were expressed in AP-1 cells (18), PMA inhibited NHE3 >50% (Fig. 6C). In contrast, NHE1 activity was
enhanced >25% (Fig. 6D, see also Ref. 19).
Moreover, the PKC antagonist chelerythrine chloride blunted the
PMA-induced inhibition of NHE5 activity (Fig. 7B),
consistent with PMA acting on NHE5 through a PKC-dependent phosphorylation process.
|
|
Regulation of rat NHE5 by PKA. Na+/H+ exchanger activity is often modified by phosphorylation-dependent events, possibly including serine/threonine sites located in the cytoplasmic carboxy terminus of the transporter (21). A number of potential phosphorylation sites are in this "regulatory" domain of rat NHE5, including PKA-dependent sites (2). To examine the role of PKA in regulating Na+/H+ exchanger activity, PS120 cells expressing NHE5 were treated with agents known to activate this pathway.
As shown in Fig. 8A and as summarized in Fig. 9, NHE5 activity was inhibited >60% by acute exposure to 10 µM forskolin, an agent that increases intracellular cAMP by activating adenylate cyclase. In contrast, there was no significant effect of the inactive forskolin analog 1,9-DDF on NHE5 activity (Fig. 8B). To verify that the effect of forskolin was mediated through cAMP, we examined the response of NHE5 to the cell-permeant cAMP analog 8-Br-cAMP. Like forskolin, 8-Br-cAMP (100 µM) inhibited NHE5 activity, nearly 70% (Fig. 8C), and the potent PKA antagonist H-89 reversed the effects of forskolin (Fig. 9B), suggesting that inhibition of NHE5 by cAMP involves a PKA-dependent process. Moreover, the PKC inhibitor chelerythrine chloride had no effect on the forskolin-induced inhibition of NHE5 (Fig. 9, compare B with A), indicating that this cAMP-dependent response did not involve the PKC signaling pathway. In contrast to cAMP, 8-Br-cGMP produced only a subtle change in NHE5 exchanger activity that was not statistically significant (Fig. 8D), suggesting that cGMP-dependent kinases do not regulate NHE5. Together, these results demonstrate that PKA activation inhibits NHE5 activity. As observed for NHE5, Fig. 9A shows that NHE3 was inhibited by forskolin and 8-Br-cAMP, whereas these agents did not have a significant effect on NHE1 activity.
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have a general understanding of the properties of Na+/H+ exchange activity in the central nervous system (5, 10, 16, 25, 26, 34, 41, 48); however, relatively little is known about the expression and function of specific NHE isoforms. NHE1, the housekeeping isoform thought to be the primary regulator of pHi in most cells, is expressed throughout the brain, and its expression is critical for normal postnatal development of the mouse central nervous system. Approximately 2 wk after birth, mice lacking expression of functional NHE1 display growth retardation, severe ataxia, and epileptic-like seizures associated with increased mortality (4, 15). In contrast, the epithelial isoform NHE3 is not present in detectable levels in the brain, with the exception of the cerebellum (9), and knocking out this gene failed to create demonstrable neurological consequences (38). Two other epithelial isoforms, NHE2 and NHE4, are expressed in the brain, although apparently to a lesser extent than NHE1 (32, 45). Targeted disruption of NHE2 failed to produce obvious neurological defects (37). More recently, a fifth plasma membrane isoform NHE5 was cloned (2, 3). NHE5 is expressed throughout the brain (3), suggesting that it may serve a specialized function in neurons. Initial characterization of the kinetic properties of human NHE5 suggests that it behaves much like the amiloride-resistant Na+/H+ exchanger activity present in hippocampal neurons (39).
To gain a better understanding of NHE5 function and its regulation, we
stably expressed this protein in cells lacking
Na+/H+ exchange. Our results indicate that many
of the basic functional properties (e.g., EIPA sensitivity and ion
selectivity) of rat NHE5 under physiological conditions (i.e., in a
high external Na+ concentration) are similar to human NHE5
(39). The amino acid sequence of rat NHE5 (2)
and its sensitivity to the Na+/H+ exchange
inhibitor EIPA are most like NHE3 (Fig. 1, see also Ref.
29). These results likely reflect the close similarity of
the NHE5 and NHE3 ion-transporting domain (62% identity) that also
contains the predicted amiloride-binding region (44). Like other members of the NHE family (with the exception of NHE4, see Ref.
13), K+ and Rb+ were
nontransportable by NHE5. The affinity of NHE5 for Na+ is
relatively low (KNa 27 mM), being
intermediate to NHE1 and NHE3 (29), 10 and 5 mM,
respectively, and NHE2 at 50 mM Na+ (49). The
Na+ affinity reported for the
Na+/H+ exchange present in rat hippocampal
neurons (KNa
23-26 mM, see Ref.
34) is comparable to rat as well as human NHE5
(KNa
19 mM, see Ref. 39),
consistent with NHE5 being expressed in this region of the brain
(2). The transport activity of rat NHE5 as a function of
the intracellular H+ concentration was similar to that
reported for other NHE isoforms (23, 29). The Hill
coefficient of rat NHE5 was ~3 for the intracellular proton
concentration with a half-maximal activity near pH 6.9. These results
are different, however, from those reported for human NHE5 in which an
apparent first-order dependence on the intracellular proton
concentration was observed with a half-maximal activity at pH ~6.43
(39). The Na+/H+ exchange in rat
hippocampal neurons also apparently displays first-order dependence on
the intracellular H+ concentration, but with a half-maximal
activity greater than pH 6.8, closer to the results of the present
study (34).
In sharp contrast to Na+/H+ exchange in rat hippocampal neurons (34) in which the pHi recovery rate from an acid load in physiological external Na+ was comparable to that in the presence of Li+, NHE5 exchanged Li+ at a very low rate compared with Na+ (Fig. 4). Other members of the NHE gene family also transport Li+ at a slower rate than Na+, although the differences in rates of translocation for Na+ and Li+ are not as dramatic (see Refs. 17 and 29, and as confirmed in the present study). This observation may relate to differences in the affinity of the Na+-binding site for Li+; that is, Li+ competition with Na+ for this site is an order of magnitude stronger in NHE5 (39) than in other NHE isoforms (29), resulting in a slower transport rate.
Together, it appears that the Na+/H+ exchanger activity described in rat hippocampal neurons cannot be easily attributed to any single NHE isoform (34). The complete insensitivity of this exchanger to amiloride (1 mM) and its derivative N,N-hexamethyleneamiloride (100 µM) contrast with the inhibition constants of these reagents and related compounds for NHE5 (2, 39). Moreover, the comparable transport rate for Na+ and Li+ on the Na+/H+ exchanger in the hippocampus is inconsistent with NHE5 expression (present study and Ref. 39). We cannot rule out the possibility that when expressed in neurons, the inhibitor sensitivity and ion selectivity of NHE5 are dramatically altered; however, this seems unlikely.
The cytoplasmic carboxy termini of NHE proteins contain elements thought to be the primary sites for regulation of transport activity, including sites for phosphorylation by serine/threonine kinases (18). The carboxy-terminal region contains the largest degree of divergence within the NHE family of proteins. This property holds true for NHE5 as well; i.e., NHE5 has <20% identity with NHE1, NHE2, or NHE4, and 26% identity with NHE3 in the carboxy termini of these proteins (2, 3). Therefore, it might be expected that the regulation of NHE5 activity by phosphorylation would be distinct, as shown for the other NHE isoforms (18, 23). Nevertheless, NHE5 responded to stimulation in a qualitatively similar manner to NHE3. NHE5 and NHE3 were inhibited to a comparable extent by PKA or PKC activation and by hypertonic shock. In contrast, NHE1 activity was not significantly affected by PKA activation but was enhanced by PKC stimulation (in the present studies) and when exposed to a hypertonic solution (see Ref. 19). Thus the regulation of NHE5 is qualitatively most like NHE3, the NHE isoform with which NHE5 shares the highest amino acid sequence identity.
The above regulation of NHE5, NHE3, and NHE1 is most likely associated with the carboxy terminus (18), possibly due to direct phosphorylation. Potential phosphorylation sites on NHE5 for PKC are located at Ser-593 and Ser-652, and PKA (and Ca2+/calmodulin-dependent kinase II) sites are predicted at Ser-649, Ser-732, Ser-855, and Ser-857 (2). Of these PKA phosphorylation sites, only the homologous site in NHE5 for Ser-649 is present in rat NHE3 Ser-661 (32). However, a mutation at this site failed to have an effect on the sensitivity of NHE3 to forskolin (21). In contrast, mutation of Ser-605 prevented phosphorylation of rat NHE3 and blunted the inhibition induced by forskolin (21). This site aligns with the PKC site located at Ser-593 in rat NHE5. It is important to note that PKA-dependent inhibition (phosphorylation) of rabbit NHE3 is thought to require the physical association of NHE3 with PKA through linker proteins (46). The PS120 cells used in previous studies apparently lacked the PKA-binding protein ezrin and the adaptor protein NHERF, thus regulation of rabbit NHE3 by PKA in this cell type requires coexpression of these proteins (50). Nevertheless, we clearly observed a dramatic inhibition of both rat NHE3 and NHE5 through a PKA-dependent process when expressed in PS120 cells. Although we did not rule out the possibility that fundamentally different mechanisms may be involved in the PKA-induced inhibition of the rat NHE3 protein, the basis for this apparent discrepancy appears to reflect differences in the PS120 clonal isolates used in our study. Figure 10 demonstrates that transcripts for both NHERF and ezrin are expressed in PS120 cells. Regardless of the mechanism(s) involved, future studies should identify the molecular machinery that regulates rat NHE5 via PKA and PKC activation and allow us to ascribe in situ function(s).
NHE5 was also regulated by changes in the cell volume in the present studies. Cell shrinkage inhibited activity, whereas swelling had no detectable effect. These results are most like those observed for NHE3, where a hypertonic solution blunted activity, and swelling was without effect (19). Conversely, NHE1 and NHE2 are activated by cell shrinkage and inhibited by swelling (19). NHE4 is coexpressed in some of the same regions of the brain (7) as NHE5 (2, 3). However, unlike NHE5, NHE4 is apparently activated by hyperosmolar conditions (8), suggesting possible overlapping, yet antagonistic, functions for these two exchangers.
In summary, we have characterized some of the kinetic and regulatory properties of rat NHE5. It is interesting to note that brain-specific NHE5 behaves qualitatively much like NHE3, an exchanger with high expression within specific regions of renal and intestinal epithelia (32). In both of these tissues, the apical membrane NHE3 plays a major role in Na+ absorption, as demonstrated in mice lacking expression of this protein (11, 38). Clearly, the function of NHE5 is quite different in neurons, where NHE5 may complement the pH housekeeping exchanger NHE1. This redundancy may be necessary in some neurons in which high metabolic activity under physiological and pathophysiological conditions results in substantial acid production. However, the regulation of NHE1 is quite different from NHE5. In contrast to NHE1 (18, 19, 23), kinase activation and cell shrinkage inhibit NHE5 activity, suggesting that these exchangers may be most active under different (patho)physiological conditions or possibly have antagonistic actions on neuronal activity. Moreover, the pK of rat NHE5 is near pH 6.9, indicating that this exchanger is active at resting pH and is poised to respond when the cell is acid loaded. At present, we can only speculate the functional significance of NHE5 activity in the brain and the response of this exchanger to the activation of protein kinases A and C and cell shrinkage. The spontaneous and epileptiform bioelectric activity of hippocampal CA3 neurons is suppressed by intracellular acidification (6). This acidification has been shown to provide protective advantage during an ischemic challenge (22). Therefore, protein kinase-dependent inhibition of NHE5 might yield protection from postischemic brain damage. Nevertheless, it is clear that Na+/H+ exchange activity is required for normal electrical activity, at least for NHE1-mediated exchanger activity. Null mutations of this latter Na+/H+ exchanger gene produce epileptic seizures (4, 15). Because of the complexity of the brain, targeted disruption of the Nhe5 gene will likely be necessary to sort out its functions.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. E. Chang and C. Bookstein for providing PS120 and PS120 cells expressing rat NHE1 and Drs. H.-V. Nguyen and J. Arreola for valuable input during the course of this study.
![]() |
FOOTNOTES |
---|
* S. Attaphitaya and K. Nehrke contributed equally to this work.
This work was supported in part by National Institute of Dental and Craniofacial Research (NIDCR) Grant DE-08921 (to J. E. Melvin). S. Attaphitaya was supported by a Dentist Scientist Award Fellowship from NIDCR (Grant DE-00159).
Address for reprint requests and other correspondence: J. E. Melvin, Center for Oral Biology, Aab Institute of Biomedical Sciences, Univ. of Rochester, Medical Center Box 611, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: james_melvin{at}urmc.rochester.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 5 October 2000; accepted in final form 22 May 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aschner, M,
Vitarella D,
Allen JW,
Conklin DR,
and
Cowan KS.
Methylmercury-induced astrocytic swelling is associated with activation of the Na+/H+ antiporter, and is fully reversed by amiloride.
Brain Res
799:
207-214,
1998[ISI][Medline].
2.
Attaphitaya, S,
Park K,
and
Melvin JE.
Molecular cloning and functional expression of a rat Na+/H+ exchanger (NHE5) highly expressed in brain.
J Biol Chem
274:
4383-4388,
1999
3.
Baird, NR,
Orlowski J,
Szabo EZ,
Zaun HC,
Schultheis PJ,
Menon AG,
and
Shull GE.
Molecular cloning, genomic organization, and functional expression of Na+/H+ exchanger isoform 5 (NHE5) from human brain.
J Biol Chem
274:
4377-4382,
1999
4.
Bell, SM,
Schreiner CM,
Schultheis PJ,
Miller ML,
Evans RL,
Vorhees CV,
Shull GE,
and
Scott WJ.
Targeted disruption of the murine Nhe1 locus induces ataxia, growth retardation, and seizures.
Am J Physiol Cell Physiol
276:
C788-C795,
1999
5.
Bevensee, MO,
Cummins TR,
Haddad GG,
Boron WF,
and
Boyarsky G.
pH regulation in single CA1 neurons acutely isolated from the hippocampi of immature and mature rats.
J Physiol (Lond)
494:
315-328,
1996[Abstract].
6.
Bonnet, U,
Leniger T,
and
Wiemann M.
Alteration of intracellular pH and activity of CA3-pyramidal cells in guinea pig hippocampal slices by inhibition of transmembrane acid extrusion.
Brain Res
872:
116-124,
2000[ISI][Medline].
7.
Bookstein, C,
Musch MW,
DePaoli A,
Xie Y,
Rabenau K,
Villereal M,
Rao MC,
and
Chang EB.
Characterization of the rat Na+/H+ exchanger isoform NHE4 and localization in rat hippocampus.
Am J Physiol Cell Physiol
271:
C1629-C1638,
1996
8.
Bookstein, C,
Musch MW,
DePaoli A,
Xie Y,
Villereal M,
Rao MC,
and
Chang EB.
A unique sodium-hydrogen exchange isoform (NHE-4) of the inner medulla of the rat kidney is induced by hyperosmolarity.
J Biol Chem
269:
29704-29709,
1994
9.
Boyarsky, G,
Ganz MB,
Sterzel RB,
and
Boron WF.
pH regulation in single glomerular mesangial cells. I. Acid extrusion in absence and presence of HCO
10.
Boyarsky, G,
Ransom B,
Schlue WR,
Davis MB,
and
Boron WF.
Intracellular pH regulation in single cultured astrocytes from rat forebrain.
Glia
8:
241-248,
1993[ISI][Medline].
11.
Brant, SR,
Yun CH,
Donowitz M,
and
Tse CM.
Cloning, tissue distribution, and functional analysis of the human Na+/H+ exchanger isoform, NHE3.
Am J Physiol Cell Physiol
269:
C198-C206,
1995
12.
Cabado, AG,
Yu FH,
Kapus A,
Lukacs G,
Grinstein S,
and
Orlowski J.
Distinct structural domains confer cAMP sensitivity and ATP dependence to the Na+/H+ exchanger NHE3 isoform.
J Biol Chem
271:
3590-3599,
1996
13.
Chambrey, R,
Achard JM,
and
Warnock DG.
Heterologous expression of rat NHE4: a highly amiloride-resistant Na+/H+ exchanger isoform.
Am J Physiol Cell Physiol
272:
C90-C98,
1997
14.
Counillon, L,
and
Pouyssegur J.
The expanding family of eucaryotic Na+/H+ exchangers.
J Biol Chem
275:
1-4,
2000
15.
Cox, GA,
Lutz CM,
Yang CL,
Biemesderfer D,
Bronson RT,
Fu A,
Aronson PS,
Noebels JL,
and
Frankel WN.
Sodium/hydrogen exchanger gene defect in slow-wave epilepsy mutant mice.
Cell
91:
139-148,
1997[ISI][Medline].
16.
Gaillard, S,
and
Dupont JL.
Ionic control of intracellular pH in rat cerebellar Purkinje cells maintained in culture.
J Physiol (Lond)
425:
71-83,
1990[Abstract].
17.
Honda, T,
Knobel SM,
Bulus NM,
and
Ghishan FK.
Kinetic characterization of a stably expressed novel Na+/H+ exchanger (NHE-2).
Biochim Biophys Acta
1150:
199-202,
1993[ISI][Medline].
18.
Kandasamy, RA,
Yu FH,
Harris R,
Boucher A,
Hanrahan JW,
and
Orlowski J.
Plasma membrane Na+/H+ exchanger isoforms (NHE-1, -2, and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways.
J Biol Chem
270:
29209-29216,
1995
19.
Kapus, A,
Grinstein S,
Wasan S,
Kandasamy R,
and
Orlowski J.
Functional characterization of three isoforms of the Na+/H+ exchanger stably expressed in Chinese hamster ovary cells. ATP dependence, osmotic sensitivity, and role in cell proliferation.
J Biol Chem
269:
23544-23552,
1994
20.
Klanke, CA,
Su YR,
Callen DF,
Wang Z,
Meneton P,
Baird N,
Kandasamy RA,
Orlowski J,
Otterud BE,
Leppert M,
Shull GE,
and
Menon AG.
Molecular cloning and physical and genetic mapping of a novel human Na+/H+ exchanger (NHE5/SLC9A5) to chromosome 16q22.1.
Genomics
25:
615-622,
1995[ISI][Medline].
21.
Kurashima, K,
Yu FH,
Cabado AG,
Szabo EZ,
Grinstein S,
and
Orlowski J.
Identification of sites required for down-regulation of Na+/H+ exchanger NHE3 activity by cAMP-dependent protein kinase. Phosphorylation-dependent and -independent mechanisms.
J Biol Chem
272:
28672-28679,
1997
22.
Kuribayashi, Y,
Itoh N,
Horikawa N,
and
Ohashi N.
SM-20220, a potent Na+/H+ exchange inhibitor, improves consciousness recovery and neurological outcome following transient cerebral ischaemia in gerbils.
J Pharm Pharmacol
52:
441-444,
2000[ISI][Medline].
23.
Levine, SA,
Montrose MH,
Tse CM,
and
Donowitz M.
Kinetics and regulation of three cloned mammalian Na+/H+ exchangers stably expressed in a fibroblast cell line.
J Biol Chem
268:
25527-25535,
1993
24.
Levine, SA,
Nath SK,
Yun CH,
Yip JW,
Montrose M,
Donowitz M,
and
Tse CM.
Separate C-terminal domains of the epithelial specific brush border Na+/H+ exchanger isoform NHE3 are involved in stimulation and inhibition by protein kinases/growth factors.
J Biol Chem
270:
13716-13725,
1995
25.
Lin, CW,
Kalaria RN,
Kroon SN,
Bae JY,
Sayre LM,
and
LaManna JC.
The amiloride-sensitive Na+/H+ exchange antiporter and control of intracellular pH in hippocampal brain slices.
Brain Res
731:
108-113,
1996[ISI][Medline].
26.
Ma, E,
and
Haddad GG.
Expression and localization of Na+/H+ exchangers in rat central nervous system.
Neuroscience
79:
591-603,
1997[ISI][Medline].
27.
Nguyen, HV,
Shull GE,
and
Melvin JE.
Muscarinic receptor-induced acidification in sublingual mucous acinar cells: loss of pH recovery in Na+-H+ exchanger-1 deficient mice.
J Physiol (Lond)
523:
139-146,
2000
28.
Numata, M,
Petrecca K,
Lake N,
and
Orlowski J.
Identification of a mitochondrial Na+/H+ exchanger.
J Biol Chem
273:
6951-6959,
1998
29.
Orlowski, J.
Heterologous expression and functional properties of amiloride high affinity (NHE-1) and low affinity (NHE-3) isoforms of the rat Na/H exchanger.
J Biol Chem
268:
16369-16377,
1993
30.
Orlowski, J,
and
Grinstein S.
Na+/H+ exchangers of mammalian cells.
J Biol Chem
272:
22373-22376,
1997
31.
Orlowski, J,
and
Kandasamy RA.
Delineation of transmembrane domains of the Na+/H+ exchanger that confer sensitivity to pharmacological antagonists.
J Biol Chem
271:
19922-19927,
1996
32.
Orlowski, J,
Kandasamy RA,
and
Shull GE.
Molecular cloning of putative members of the Na/H exchanger gene family. cDNA cloning, deduced amino acid sequence, and mRNA tissue expression of the rat Na/H exchanger NHE-1 and two structurally related proteins.
J Biol Chem
267:
9331-9339,
1992
33.
Park, K,
Olschowka JA,
Richardson LA,
Bookstein C,
Chang EB,
and
Melvin JE.
Expression of multiple Na+/H+ exchanger isoforms in rat parotid acinar and ductal cells.
Am J Physiol Gastrointest Liver Physiol
276:
G470-G478,
1999
34.
Raley-Susman, KM,
Cragoe EJ, Jr,
Sapolsky RM,
and
Kopito RR.
Regulation of intracellular pH in cultured hippocampal neurons by an amiloride-insensitive Na+/H+ exchanger.
J Biol Chem
266:
2739-2745,
1991
35.
Roos, A,
and
Boron WF.
Intracellular pH.
Physiol Rev
61:
296-434,
1981
36.
Sardet, C,
Franchi A,
and
Pouyssegur J.
Molecular cloning, primary structure, and expression of the human growth factor-activatable Na+/H+ antiporter.
Cell
56:
271-280,
1989[ISI][Medline].
37.
Schultheis, PJ,
Clarke LL,
Meneton P,
Harline M,
Boivin GP,
Stemmermann G,
Duffy JJ,
Doetschman T,
Miller ML,
and
Shull GE.
Targeted disruption of the murine Na+/H+ exchanger isoform 2 gene causes reduced viability of gastric parietal cells and loss of net acid secretion.
J Clin Invest
101:
1243-1253,
1998
38.
Schultheis, PJ,
Clarke LL,
Meneton P,
Miller ML,
Soleimani M,
Gawenis LR,
Riddle TM,
Duffy JJ,
Doetschman T,
Wang T,
Giebisch G,
Aronson PS,
Lorenz JN,
and
Shull GE.
Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+ exchanger.
Nat Genet
19:
282-285,
1998[ISI][Medline].
39.
Szabo, EZ,
Numata M,
Shull GE,
and
Orlowski J.
Kinetic and pharmacological properties of human brain Na+/H+ exchanger isoform 5 stably expressed in Chinese hamster ovary cells.
J Biol Chem
275:
6302-6307,
2000
40.
Thomas, JA,
Buchsbaum RN,
Zimniak A,
and
Racker E.
Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ.
Biochemistry
18:
2210-2218,
1979[ISI][Medline].
41.
Tolkovsky, AM,
and
Richards CD.
Na+/H+ exchange is the major mechanism of pH regulation in cultured sympathetic neurons: measurements in single cell bodies and neurites using a fluorescent pH indicator.
Neuroscience
22:
1093-1102,
1987[ISI][Medline].
42.
Tse, CM,
Levine SA,
Yun CH,
Brant SR,
Pouyssegur J,
Montrose MH,
and
Donowitz M.
Functional characteristics of a cloned epithelial Na+/H+ exchanger (NHE3): resistance to amiloride and inhibition by protein kinase C.
Proc Natl Acad Sci USA
90:
9110-9114,
1993[Abstract].
43.
Wakabayashi, S,
Fafournoux P,
Sardet C,
and
Pouyssegur J.
The Na+/H+ antiporter cytoplasmic domain mediates growth factor signals and controls "H+-sensing".
Proc Natl Acad Sci USA
89:
2424-2428,
1992[Abstract].
44.
Wakabayashi, S,
Shigekawa M,
and
Pouyssegur J.
Molecular physiology of vertebrate Na+/H+ exchangers.
Physiol Rev
77:
51-74,
1997
45.
Wang, Z,
Orlowski J,
and
Shull GE.
Primary structure and functional expression of a novel gastrointestinal isoform of the rat Na/H exchanger.
J Biol Chem
268:
11925-11928,
1993
46.
Weinman, EJ,
Steplock D,
Tate K,
Hall RA,
Spurney RF,
and
Shenolikar S.
Structure-function of recombinant Na/H exchanger regulatory factor (NHE-RF).
J Clin Invest
101:
2199-2206,
1998
47.
Wiederkehr, MR,
Zhao H,
and
Moe OW.
Acute regulation of Na/H exchanger NHE3 activity by protein kinase C: role of NHE3 phosphorylation.
Am J Physiol Cell Physiol
276:
C1205-C1217,
1999
48.
Yao, H,
Ma E,
Gu XQ,
and
Haddad GG.
Intracellular pH regulation of CA1 neurons in Na+/H+ isoform 1 mutant mice.
J Clin Invest
104:
637-645,
1999
49.
Yu, FH,
Shull GE,
and
Orlowski J.
Functional properties of the rat Na/H exchanger NHE-2 isoform expressed in Na/H exchanger-deficient Chinese hamster ovary cells.
J Biol Chem
268:
25536-25541,
1993
50.
Yun, CH,
Oh S,
Zizak M,
Steplock D,
Tsao S,
Tse CM,
Weinman EJ,
and
Donowitz M.
cAMP-mediated inhibition of the epithelial brush border Na+/H+ exchanger, NHE3, requires an associated regulatory protein.
Proc Natl Acad Sci USA
94:
3010-3015,
1997
51.
Yun, CH,
Tse CM,
and
Donowitz M.
Chimeric Na+/H+ exchangers: an epithelial membrane-bound N-terminal domain requires an epithelial cytoplasmic C-terminal domain for regulation by protein kinases.
Proc Natl Acad Sci USA
92:
10723-10727,
1995[Abstract].
52.
Yun, CH,
Tse CM,
Nath S,
Levine SL,
and
Donowitz M.
Structure/function studies of mammalian Na-H exchangers-an update.
J Physiol
482:
1S-6S,
1995[Medline].
53.
Zhao, H,
Wiederkehr MR,
Fan L,
Collazo RL,
Crowder LA,
and
Moe OW.
Acute inhibition of Na/H exchanger NHE-3 by cAMP. Role of protein kinase A and NHE-3 phosphoserines 552 and 605.
J Biol Chem
274:
3978-3987,
1999