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J Biol Chem, Vol. 274, Issue 7, 4383-4388, February 12, 1999
From the We report here the cloning, primary structure,
heterologous expression, tissue distribution, and localization of a
cDNA encoding rat NHE5, a fifth member of the mammalian plasma
membrane Na+/H+ exchanger (NHE) gene
family. The full-length open reading frame as well as 34 nucleotides of
5'-untranslated and 1443 nucleotides of 3'-untranslated sequences were
obtained using a polymerase chain reaction strategy involving reverse
transcription-polymerase chain reaction and 5'/3'-rapid amplification
of cDNA ends. The NHE5 cDNA encodes a protein of 898 amino
acids with a calculated Mr of 99,044 and is
predicted to contain 11-13 transmembrane domains. An amino acid
comparison of the coding region of rat NHE5 reveals 95% identity with
human NHE5. Northern hybridization analysis showed that high level
expression of NHE5 mRNA is restricted to brain. Transfection of the
coding region of rat NHE5 into NHE-deficient PS120 cells resulted in
Na+/H+ exchange activity that was relatively
insensitive to the amiloride analogue,
5-(N-ethyl-N-isopropyl) amiloride, with a
half-maximal inhibitory concentration (IC50) of 1.53 ± 0.25 µM. In situ hybridization of rat
brain sections revealed significant NHE5 mRNA levels in the dentate
gyrus with lower levels observed in the hippocampus and cerebral
cortex. These results suggest a specialized role for this fifth NHE
isoform in neuronal tissues.
Na+/H+ exchangers
(NHE)1 comprise a family of
integral plasma membrane proteins involved in a variety of
physiological processes such as pH regulation, cellular
differentiation, cell volume control, and electrolyte transport
(reviewed in Refs. 1-3). Since the initial cloning of the human growth
factor activable Na+/H+ exchanger, NHE1 (4),
four additional plasma membrane NHE isoforms have been reported (5-7).
Recently a human mitochondrial isoform, NHE6, has also been cloned (8).
Characterization of the individual NHE isoforms has revealed
differences in exchange kinetics, pHi sensitivity, mode of
regulation, cellular localization, and tissue expression (1-3). As a
result of this diversity, specialization of function has been
postulated for the different NHE isoforms and, in some cases, confirmed
in null-mutant mice (9-11).
Rat hippocampal neurons express an amiloride-insensitive
Na+/H+ exchanger, the molecular identity of
which is unknown (12). Pharmacological characterization of the
different NHE isoforms indicates that rat NHE1 and NHE2 are very
sensitive to amiloride and, therefore, not the hippocampal
Na+/H+ exchanger (5, 6). In contrast, rat NHE3
and NHE4 are relatively insensitive to amiloride (5, 13, 14); however,
little NHE3 mRNA is expressed in rat brain (5), and NHE4, unlike
the Na+/H+ exchanger expressed in hippocampal
neurons (12), is inhibited by high concentrations of amiloride (13,
14). Consequently, neither NHE3 nor NHE4 are likely to represent the
activity of an amiloride-insensitive Na+/H+
exchanger previously described in hippocampal neurons (12). The partial
coding sequence for a fifth isoform primarily expressed in brain, NHE5,
is most closely related to NHE3 (7), and therefore NHE5 might also be
expected to express amiloride resistance. To investigate further the
properties of this putative brain-specific Na+/H+ exchanger, we cloned the rat NHE5
cDNA and determined its primary structure, tissue distribution, and
localization. NHE5 encodes for a functional
Na+/H+ exchanger that is resistant to the
amiloride derivative EIPA and is highly expressed in the dentate gyrus.
Cloning of the Rat NHE5 cDNA--
Total RNA was prepared
from whole brain of male Wistar rats (150-200 g) using TRIzol reagent
(Life Technologies, Inc.). RNA was reverse-transcribed to cDNA
using oligo(dT) and random hexamer primers according to the
manufacturer's recommendations (1st Strand cDNA
Synthesis kit, CLONTECH, Palo Alto, CA). Rat brain cDNA was then used in subsequent RT-PCR reactions. 5'- and 3'-RACE was performed using adapter-ligated rat brain Marathon-Ready cDNAs (CLONTECH, Palo Alto, CA). RT-PCR and RACE
amplification products were subcloned into the pCR 2.1 vector
(Invitrogen, San Diego, CA). DNA sequence analysis of both strands was
performed manually using the T7 Sequenase 7-deaza-dGTP sequencing kit
(Amersham Pharmacia Biotech). Sequence analysis and alignment were
performed using MacVector software (International Biotechnologies Inc.,
New Haven, CT).
Four overlapping partial cDNA clones of rat NHE5 (rNHE5a
-rNHE5d) provided the complete nucleotide sequence of rat NHE5 (rNHE5) cDNA, including 5'- and 3'-untranslated regions (UTR). We then amplified and cloned the coding region of rNHE5 by RT-PCR. The rNHE5
open reading frame was placed into an eukaryotic expression vector,
pCMV/SEAP (Tropix, Bedford, MA), generating the expression construct
pCMV/rNHE5 used in functional studies. The following is a description
detailing the procedures and primers used to amplify the above clones.
The initial 384-bp clone (rNHE5a) was amplified using human NHE5
primers based on previously published partial coding sequences (7). The
primers used were 5'-TGCTGGGCCTGGTGCTA-3', corresponding to codons
82-87, and 5'-ACTCGCCAAAGACGATGAT-3', complementary to codons 204-209
of human NHE5 (7). PCR amplification was performed for 30 cycles using
Amplitaq DNA polymerase (Perkin-Elmer) on a DNA thermal cycler (PTC-200
Peltier Thermal Cycler, MJ Research, Watertown, MA) with each cycle set
to 94 °C for 30 s, 64 °C for 30 s, and 72 °C for 2 min. A 5-µl aliquot of the first-round PCR reaction mixture was
re-amplified using the same PCR conditions.
The second 592-bp clone (rNHE5b) corresponded to the 5' end of rat NHE5
(roughly the first six transmembrane domains) and included 34 bases of
the 5'-untranslated region. This clone was amplified via 5'-RACE using
an AP1 primer (5'-CCATCCTAATACGACTCACTATAGGGC-3') complementary to the
adapter region and a rat NHE5-specific primer (5'-CACCGCTGAGATGAGGCTTCCAAAGA-3') complementary to codons 178-186. PCR amplification was performed using CLONTECH's
Advantage cDNA Polymerase Mix as follows: 94 °C for 1 min
followed by 15 cycles at 94 °C for 30 s then 72 °C for 4 min, 5 cycles at 94 °C for 30 s then 70 °C for 4 min, and 20 cycles at 94 °C for 30 s then 68 °C for 4 min. A 1-µl
aliquot of the first-round PCR reaction mixture was re-amplified under
the same PCR conditions.
The third 2103-bp clone (rNHE5c) spanning the majority of the coding
region was generated via RT-PCR using a rat NHE5-specific primer
(5'-GCCGGGGACCTTCTTCCTTTTCCTG-3') corresponding to codons 106-113 and
a primer (5'-GGGGACGCTAGGCTCTCCAGGGATGA-3') complementary to codons
796-804 of human NHE5.2 PCR
reactions were the same as described above for rNHE5a.
The fourth 1866-bp clone (rNHE5d) corresponded to the remainder of the
coding region and the entire 3'-untranslated region. This clone was
attained through 3'-RACE using a rat NHE5-specific primer
(5'-TGCGCGGAGAAAGAGCTACCCTGGAA-3') corresponding to codons 759-767 and
AP1 as the adapter primer. PCR conditions were the same as described
for rNHE5b.
The rNHE5 expression clone containing 34 bp of 5'-UTR, the entire
coding region, and 7 bp of 3'-UTR was amplified by RT-PCR using high
fidelity Pfu polymerase (Stratagene, La Jolla, CA), rat
brain cDNA as template, and rat NHE5-specific primers. The primers
used were 5'-acgaagcttGCCGGCGGCCGTGCAGTGCCCGGAG-3', corresponding to
nts -34 to -10, and
5'-actctagaCCCTGGGCTACAGCCTGCCTCCTCTGTT-3', complementary to nts
2677-2704. Lowercase letters represent HindIII and
XbaI restriction sites flanking each primer, placed to
facilitate ligation into the eukaryotic expression vector.
Transfection and Stable Expression of rNHE5--
The rNHE5 PCR
product was digested by HindIII and XbaI
restriction enzymes and then subcloned into pCMV/SEAP previously
digested with HindIII and XbaI. The orientation
and coding region were verified by sequencing. NHE-deficient PS120
cells were maintained in Dulbecco's modified Eagle's medium (Life
Technologies, Inc.) supplemented with 10% fetal bovine serum,
L-glutamine, and penicillin (50 units/ml)/streptomycin (50 µg/ml) and selected by the H+-suicide technique as
described previously (15). To establish stable expression of rat NHE5,
the NHE-deficient PS120 cells were transfected with 20 µg of
pCMV/rNHE5 by the CaPO4-DNA coprecipitation method (16).
Cells were selected for stable expression with G418 (1000 units/ml,
Life Technologies, Inc.) and further selected for expression of
Na+/H+ exchanger activity by the
"H+ killing" method (17). NHE5 transfected cells were
maintained in medium containing G418, and the "H+
killing" procedure was repeated every 4-5 days.
Intracellular pH Measurements--
Intracellular pH was
monitored using the pH-sensitive dye SNARF-1 (Molecular Probes, Eugene,
OR) as described previously (18). Cells plated on glass coverslips
mounted on the microscope stage of an Ultima confocal laser cytometer
(Meridian Instruments, Okemos, MI) were acid-loaded using the
NH4Cl pre-pulse technique (19). 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 (Na+ was replaced by
N-methyl-D-glucamine) to produce an acid load. Approximately 3 min later, extracellular Na+ was restored
to initiate Na+/H+ exchanger-mediated
intracellular pH recovery. The physiological salt solution contained
(in mM): NaCl (135), KCl (5.4),
KH2PO4 (0.4), NaH2PO4
(0.33), glucose (10), Hepes (20), CaCl2 (1.2), MgSO4 (0.8), pH 7.4. Recovery rates of the fluorescence
traces were determined from the initial linear portion following the re-addition of extracellular Na+ and expressed as mean ± S.E. Calibration of the intracellular pH signal was accomplished by
the high potassium-nigericin technique (20).
Northern Hybridization Analysis--
Northern analysis was
performed using a rat multiple tissue Northern blot containing
approximately 2 µg of poly (A)+ RNA per lane from eight
different tissues (CLONTECH, Palo Alto, CA). The
membrane was prehybridized at 60 °C in 5× SSPE, 2× Denhardt's reagent, 0.5% SDS, 100 µg/ml salmon sperm DNA, and 50 µg/ml tRNA. The 32P-labeled rat NHE5 probe was added, and hybridization
was performed in the same solution at 60 °C for 20 h. The
384-bp cDNA probe included sequence spanning codons 84-211 of rat
NHE5. The membrane was washed 4 times in 2× SSC containing 0.05% SDS
at room temperature, 2 times in 2× SSC containing 0.1% SDS at room
temperature, 1 time in 0.1× SSC containing 0.1% SDS at 68 °C and
was exposed to film for 8 days at In Situ Hybridization--
In situ hybridization was
used to investigate the possibility that rNHE5 may represent the
amiloride-insensitive NHE variant previously reported by Raley-Susman
et al. (12) in rat hippocampal neurons. Male Wistar strain
rats (150-250 g) were anesthetized by intraperitoneal injection of
ketamine (100 mg/kg body weight) and then perfused with 4%
paraformaldehyde, pH 7.4. The isolated whole brain was placed in 4%
paraformaldehyde overnight and then transferred to cryoprotectant
consisting of 30% sucrose in 0.067 M phosphate-buffered
saline for 2 days. Frozen sections containing the hippocampus were cut
at 18 µm on a cryostat (HistoStat microtome, Scientific Instruments).
Labeled antisense and sense RNA probes were prepared by in
vitro transcription (SureSite II System kit, Novagen) of
pCR2.1/rNHE5d using T7 RNA polymerase in the presence of
35S-UTP. Hybridizations were performed under high
stringency conditions as described previously (21). Autoradiography of
slides was initially done for 5 days with Kodak Biomax MR film (Eastman
Kodak Co.). Slides were subsequently dipped in NT2B (Kodak) emulsion (1:1 emulsion to glycerol) and stored at 4 °C for 2 weeks before developing. Hematoxylin and eosin counterstaining was performed on
selected slides.
PCR Cloning of a Rat Brain NHE5 cDNA--
Human NHE5 is highly
expressed in brain tissue (7). We amplified a 384-bp product from rat
brain cDNA, rNHE5a, using primers based on the partial coding
sequences provided in this earlier report. The nucleotide sequence and
translation of rat NHE5 is depicted in Fig.
1. Nucleotide sequence analysis of rNHE5a
yielded >90% identity with human NHE5 suggesting that this cDNA
was the rat homolog of human NHE5. We subsequently used sequence
information gained from rNHE5a to design primers for 5'-RACE to amplify
the amino-terminal coding sequence, clone rNHE5b. The 592-bp, rNHE5b clone included 34 bp of 5'-UTR containing a purine at the
A third clone, rNHE5c, was derived by RT-PCR amplification using a rat
NHE5-specific sense primer and a human NHE5 antisense primer.2 Amplification with these primers produced a
2103-bp product corresponding to the majority of the coding region of
rat NHE5. The remaining 3'-coding region and the 3'-UTR were then
obtained through 3'-RACE. This fourth clone, rNHE5d, was 1866 bp and
included 423 bp of coding region upstream of the stop codon and 1443 bp
of 3'-UTR. A consensus AATAAA polyadenylation signal preceded by 20 nucleotides the 3' poly(A) tract, indicating an intact 3' terminus
(double underline in Fig. 1). Thus, the overlapping clones,
rNHE5a-d, provided sequence information encoding the entire rat NHE5 cDNA.
Deduced Amino Acid Sequence of NHE5--
The amino acid
sequence alignment of rat and human NHE5 is shown in Fig.
2. The 898-amino acid sequence of rat
NHE5 is 95% identical to human NHE5. In comparison with other
isoforms, rat NHE5 is most closely related to rat NHE3 (51% identity).
Highest similarity between NHE5 and NHE3 is in the amino-terminal
transmembrane domain (62%), whereas the cytoplasmic carboxyl-terminal
domain is less similar (31%). The amino acid identity to other rat NHE isoforms is considerably less, with 39% identity to rat NHE1 and NHE2,
and 31% identity to rat NHE4.
Hydropathy analysis, using the algorithm of Kyte and Doolittle (23),
predicts that the amino terminus contains 11-13 hydrophobic domains,
with an extensive carboxyl-terminal hydrophilic domain. This general
structure is consistent with the predicted topology of other NHE
isoforms. Three potential N-glycosylation sites are present
at Asn-201, Asn-321, and Asn-794; however, only Asn-321 is common to
the other potential N-glycosylation sites observed in the
different rat NHE isoforms (5, 6). One mode of regulation for rat
Na+/H+ exchanger activity is mediated through
phosphorylation of the cytoplasmic domain (24). A number of potential
phosphorylation sites are present in rNHE5 including protein kinase C
phosphorylation sites located at Ser-593 and Ser-652, and
cAMP-dependent kinase and
Ca+/calmodulin-dependent kinase II sites found
at Ser-649, Ser-732, Ser-855, and Ser-857. All of these potential
regulatory sites are also present in human NHE5 (25).
Tissue Distribution of NHE5--
Northern hybridization
analysis of a rat multiple tissue Northern blot under high stringency
conditions indicated that size as well as tissue distribution of rat
NHE5 mRNA paralleled that reported for human NHE5 (7). The highest
levels of mRNA were observed in brain (Fig.
3). The dominant mRNA was ~4.4 kb,
although as observed for human NHE5, larger bands ranging between 9 and 9.5 kb were also detected. These larger transcripts may represent retained intron sequences as observed by Baird et al. (25)
for human NHE5. Long exposure times were required to detect NHE5
mRNA expression in spleen, lung, skeletal muscle, and testis.
Localization of NHE5 in Rat Brain--
The high levels of NHE5
mRNA present in rat brain may correlate with a unique
Na+/H+ exchange activity expressed in
hippocampal neurons (12). To explore this possibility, in
situ hybridization of rat brain sections was performed. Probes
were prepared by in vitro transcription of pCR2.1/rNHE5d.
The rNHE5d clone corresponds to the 3'-untranslated region of rat NHE5
that has no similarity to other cloned NHE isoforms. Fig.
4A shows that NHE5 mRNA
was strongly expressed in the dentate gyrus, with lower levels of
expression seen in the CA1 field of the hippocampus and in the cerebral
cortex. In comparison, no significant hybridization signals were
observed when adjacent brain slices were probed with the sense probe
(Fig. 4B). Higher magnification within the dentate gyrus
(see arrow in Fig. 4A) revealed that NHE5
mRNA expression localized to the region containing the cell
bodies of neurons (Fig. 4, C and D).
Functional Expression of rNHE5 in NHE-deficient PS120
Cells--
To determine whether rNHE5 is a functional
Na+/H+ exchanger, RT-PCR was used to obtain a
clone containing the entire coding sequence. The rNHE5 clone contained
34 bp of the 5'-UTR, the coding region, and 7 bp of 3'-UTR. Underlined
nucleotides in Fig. 1 indicate the position of the sense and antisense
primers used for amplification. Intracellular pH recovery from an acid
load was not observed in nontransfected control cells (Fig.
5). In contrast, an intracellular pH
recovery was activated in transfected cells following the addition of
Na+ at the time indicated by the arrow. The
Na+-dependent pH recovery observed in rNHE5
expressing cells was inhibited by the amiloride analogue EIPA (Fig.
6). Like NHE3, rat NHE5 was relatively
resistant to inhibition by this compound. The half-maximal
concentration for inhibition of rat NHE5 (1.53 ± 0.25 µM) was comparable to the previously reported (19) EIPA sensitivity of rat NHE3 (2.4 µM).
In this report we describe the molecular cloning and complete
nucleotide sequence of a rat cDNA encoding the fifth member of the
mammalian plasma membrane NHE family, NHE5. The cDNA contains 34 and 1443 bp of 5' and 3' untranslated sequences, respectively. The 2694 bp open reading frame encodes a protein of 898 amino acids with a
predicted Mr of 99,044, making NHE5 the largest
member of the Na+/H+ exchanger gene family (5,
6).
The data presented in Fig. 3 demonstrated that high level expression of
NHE5 mRNA was restricted to rat brain, comparable to the results
described previously by Klanke et al. (7) for human tissues.
Na+/H+ exchanger activity has been described in
various neuronal tissues (26-28), including an amiloride-insensitive
Na+/H+ exchanger in cultured hippocampal
neurons (12). Neuronal expression of the different NHE isoforms has
been demonstrated for the ubiquitous housekeeping isoform, NHE1, as
well as the epithelial isoforms NHE2 and NHE4, although the latter two
isoforms are apparently expressed to a lesser extent (5, 6). We found
by in situ hybridization that NHE5 mRNA was most
abundant in the dentate gyrus. Lesser expression of NHE5 was observed
in the cerebral cortex and the CA1 field of the hippocampus. In
agreement with these observations, Baird et al. (25)
detected human NHE5 mRNA in hippocampus and several other regions
within the brain. Bookstein et al. (13) previously showed by
in situ hybridization that rat NHE4 is localized to the CA
fields of the hippocampus. Coexpression of rat NHE4 and NHE5 in the CA1
field of the hippocampus suggests possible overlap of function.
However, NHE4 is uniquely activated by hyperosmolar conditions (13),
suggesting that it may have a highly specialized function in this
region of the brain.
Fig. 6 shows that expression of rat NHE5 resulted in
Na+/H+ exchanger activity that was relatively
resistant to the amiloride derivative EIPA. Similarly, human NHE5
mediates amiloride-sensitive Na+/H+ exchange
(25). Thus, amiloride and its derivatives inhibit all five members of
the plasma membrane Na+/H+ exchanger gene
family. The EIPA sensitivity of rat NHE5 was most like rat NHE3 (19),
as might be predicted from the structural similarity of the ion
transporting domains of these two proteins (62% identity). In
contrast, Raley-Susman et al. (12) found that the
Na+/H+ exchanger activity expressed in
hippocampal neurons was not inhibited by amiloride or the amiloride
derivative 5-(N,N-hexamethylene)amiloride. The simplest
interpretation of this lack of sensitivity to amiloride is that NHE5
does not mediate the Na+/H+ exchanger activity
that was detected in cultured hippocampal neurons. Alternatively, in
hippocampal neurons the amiloride sensitivity of NHE5 may be modified
by a mechanism not present in fibroblasts (Fig. 6).
In summary, we have identified the rat homologue of the human
Na+/H+ exchanger, NHE5. The deduced amino acid
sequence and predicted membrane organization is similar to other
members of the plasma membrane Na+/H+ exchanger
gene family, being most similar to the epithelial
Na+/H+ exchanger, NHE3. Its pharmacological
properties appear to be most like NHE3 as well. In contrast to NHE3
expression, NHE5 mRNA is restricted almost exclusively to the brain
suggesting that this isoform may perform a specialized role in neuronal
tissues. Investigations are currently under way to test whether the
sequence similarity between NHE3 and NHE5 correlates with comparable
functional properties.
We thank Drs. Nancy Baird and Gary Shull for
helpful discussions during the course of this work and sharing the
sequence of human NHE5; Dr. John Olschowka for assistance in perfusion
of rats and histological identification of neuronal structures; and Linda Richardson for technical assistance in the expression studies. We
also thank Drs. Linda Callahan and Paul Coleman for allowing use of
histological equipment and suggestions for in situ hybridization.
*
This work was supported in part by National Institutes of
Health Grant DE08921 (to J. E. M.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF100172.
¶
Supported by NIDR Dentist Scientist Fellowship Award DE00159
from the National Institutes of Health.
The abbreviations used are:
NHE, Na+/H+ exchanger; NHE1-6, Na+/H+ exchanger isoform 1-6; kb, kilobase; EIPA, 5-(N-ethyl-N-isopropyl)amiloride; RT-PCR, reverse transcription-polymerase chain reaction; RACE, rapid
amplification of cDNA ends; CA, cavi amnoni; nt, nucleotide; bp, base pair; kb, kilobase pair; UTR, untranslated regions.
2
Nancy Baird and Gary E. Shull, personal communication.
Molecular Cloning and Functional Expression of a Rat
Na+/H+ Exchanger (NHE5) Highly Expressed in
Brain*
§¶,, and
Center for Oral Biology and the
§ Department of Neurobiology and Anatomy, University of
Rochester School of Medicine and Dentistry,
Rochester, New York 14642
ABSTRACT
Top
Abstract
Introduction
References
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
86 °C. The same blot was then
stripped and hybridized with a control rat 
-actin probe.
RESULTS
3 position as described for numerous eukaryotic genes by Kozak (22). Together, rNHE5a and rNHE5b provided sequence encoding the first six
transmembrane domains.
View larger version (80K):
[in a new window]
Fig. 1.
Nucleotide and deduced amino acid sequence of
rNHE5. Nucleotides are numbered on the right
and amino acids are numbered below the sequence.
Underlined nucleotides from 34 to 10 and 2677 to 2704 represent sense and antisense primers used for amplification of the
cDNA forming the expression construct pCMV/rNHE5. The consensus
polyadenylation signal is indicated by the double
underline.
View larger version (76K):
[in a new window]
Fig. 2.
Amino acid sequence alignment of rat NHE5 and
human NHE5. The amino acid sequence of rat NHE5 is compared with
human NHE5 (24). Alignment of rat and human NHE5 reveals 95% amino
acid identity. Identical amino acids are boxed.
View larger version (93K):
[in a new window]
Fig. 3.
Tissue distribution of rNHE5 mRNA.
Northern blot containing approximately 2 µg of poly(A)+
RNA per lane from eight different tissues was probed under high
stringency conditions with a 384-bp cDNA probe. The probe contained
specific sequence corresponding to codons 84-211 of rat NHE5. The film
was exposed for 8 days. A 4.4-kb mRNA was detected at the highest
levels in brain.
View larger version (124K):
[in a new window]
Fig. 4.
Localization of rNHE5 by in situ
hybridization. Alternating sections of rat brain were
hybridized with antisense and sense RNA probes containing the 3'-UTR
and 423 bp upstream of the stop codon of rat NHE5. A,
highest rNHE5 signal is localized to the dentate gyrus (DG),
with lower levels detected in the CA1 field of the hippocampus
(Hip) and the cerebral cortex (CT). B,
an adjacent section hybridized with the sense probe. C,
detailed view of region indicated by the open arrow in
A showing hematoxylin- and eosin-stained neuronal cell
bodies. Samples were viewed and analyzed on a Zeiss Axioplan microscope
using a 20 × neofluor objective (Carl Ziess, Inc., Germany).
D, same region as in C detailing silver grains deposited by
radioactive probe on neuronal cell bodies under dark-field
illumination.
View larger version (27K):
[in a new window]
Fig. 5.
Na+-dependent
recovery of the intracellular pH in cells expressing rNHE5.
SNARF-1 loaded cells were acidified using the
NH4+ pre-pulse technique. Recovery from
an acid load was initiated by the re-addition of external
Na+ at time indicated by the arrow. Shown are
typical results from rNHE5-expressing cells ( 
) and non-transfected
(
) control cells. Each data point represents the mean ± S.E.
of n > 30 cells.
View larger version (11K):
[in a new window]
Fig. 6.
EIPA sensitivity of rNHE5. The
concentration dependence of EIPA inhibition was tested in a range from
100 nM to 50 µM EIPA. The continuous
line represents a sigmoidal fit of the data providing a
half-maximal inhibitory concentration (IC50) of 1.53 ± 0.253 µM. Each data point represents the
mean percent inhibition, relative to controls, of n > 20 different cells from three separate experiments for the various EIPA
concentrations. Data shown are mean ± S.E. In all cases the
standard error was less than 5% of the mean.
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Center for Oral
Biology, Box 611, University of Rochester School of Medicine and Dentistry, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-8705; Fax: 716-473-2679; E-mail: james_melvin{at}urmc.rochester.edu.
REFERENCES
Top
Abstract
Introduction
References
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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 A. K. Pullikuth, K. Aimanova, W. Kang'ethe, H. R. Sanders, and S. S. Gill Molecular characterization of sodium/proton exchanger 3 (NHE3) from the yellow fever vector, Aedes aegypti J. Exp. Biol., September 15, 2006; 209(18): 3529 - 3544. [Abstract] [Full Text] [PDF]
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E. J. Rios, M. Fallon, J. Wang, and L. A. Shimoda Chronic hypoxia elevates intracellular pH and activates Na+/H+ exchange in pulmonary arterial smooth muscle cells Am J Physiol Lung Cell Mol Physiol, November 1, 2005; 289(5): L867 - L874. [Abstract] [Full Text] [PDF]
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 H. Xu, R. Chen, and F. K. Ghishan Subcloning, localization, and expression of the rat intestinal sodium-hydrogen exchanger isoform 8 Am J Physiol Gastrointest Liver Physiol, July 1, 2005; 289(1): G36 - G41. [Abstract] [Full Text] [PDF]
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C. L. Brett, M. Donowitz, and R. Rao Evolutionary origins of eukaryotic sodium/proton exchangers Am J Physiol Cell Physiol, February 1, 2005; 288(2): C223 - C239. [Abstract] [Full Text] [PDF]
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N. Nakamura, S. Tanaka, Y. Teko, K. Mitsui, and H. Kanazawa Four Na+/H+ Exchanger Isoforms Are Distributed to Golgi and Post-Golgi Compartments and Are Involved in Organelle pH Regulation J. Biol. Chem., January 14, 2005; 280(2): 1561 - 1572. [Abstract] [Full Text] [PDF]
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 C. Sheldon, A. Diarra, Y. M. Cheng, and J. Church Sodium Influx Pathways during and after Anoxia in Rat Hippocampal Neurons J. Neurosci., December 8, 2004; 24(49): 11057 - 11069. [Abstract] [Full Text] [PDF]
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D. A. Brown, J. E. Melvin, and D. I. Yule Critical role for NHE1 in intracellular pH regulation in pancreatic acinar cells Am J Physiol Gastrointest Liver Physiol, November 1, 2003; 285(5): G804 - G812. [Abstract] [Full Text] [PDF]
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 Y. Wang, J. W. Meyer, M. Ashraf, and G. E. Shull Mice With a Null Mutation in the NHE1 Na+-H+ Exchanger Are Resistant to Cardiac Ischemia-Reperfusion Injury Circ. Res., October 17, 2003; 93(8): 776 - 782. [Abstract] [Full Text] [PDF]
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M. CHESLER Regulation and Modulation of pH in the Brain Physiol Rev, October 1, 2003; 83(4): 1183 - 1221. [Abstract] [Full Text] [PDF]
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K. Szaszi, A. Paulsen, E. Z. Szabo, M. Numata, S. Grinstein, and J. Orlowski Clathrin-mediated Endocytosis and Recycling of the Neuron-specific Na+/H+ Exchanger NHE5 Isoform. REGULATION BY PHOSPHATIDYLINOSITOL 3'-KINASE AND THE ACTIN CYTOSKELETON J. Biol. Chem., November 1, 2002; 277(45): 42623 - 42632. [Abstract] [Full Text] [PDF]
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K. Nehrke and J. E. Melvin The NHX Family of Na+-H+ Exchangers in Caenorhabditis elegans J. Biol. Chem., August 2, 2002; 277(32): 29036 - 29044. [Abstract] [Full Text] [PDF]
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 C. V. Rieder and L. Fliegel Developmental regulation of Na+/H+ exchanger expression in fetal and neonatal mice Am J Physiol Heart Circ Physiol, July 1, 2002; 283(1): H273 - H283. [Abstract] [Full Text] [PDF]
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S. Attaphitaya, K. Nehrke, and J. E. Melvin Acute inhibition of brain-specific Na+/H+ exchanger isoform 5 by protein kinases A and C and cell shrinkage Am J Physiol Cell Physiol, October 1, 2001; 281(4): C1146 - C1157. [Abstract] [Full Text] [PDF]
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H. Yao, X.-Q. Gu, R. M. Douglas, and G. G. Haddad Role of Na+/H+ exchanger during O2 deprivation in mouse CA1 neurons Am J Physiol Cell Physiol, October 1, 2001; 281(4): C1205 - C1210. [Abstract] [Full Text] [PDF]
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 R. Chambrey, P. L. St. John, D. Eladari, F. Quentin, D. G. Warnock, D. R. Abrahamson, R.-A. Podevin, and M. Paillard Localization and functional characterization of Na+/H+ exchanger isoform NHE4 in rat thick ascending limbs Am J Physiol Renal Physiol, October 1, 2001; 281(4): F707 - F717. [Abstract] [Full Text] [PDF]
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C. Bagnis, M. Marsolais, D. Biemesderfer, R. Laprade, and S. Breton Na+/H+-exchange activity and immunolocalization of NHE3 in rat epididymis Am J Physiol Renal Physiol, March 1, 2001; 280(3): F426 - F436. [Abstract] [Full Text] [PDF]
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M. E. Giannakou and J. A. T. Dow Characterization of the Drosophila melanogaster alkali-metal/proton exchanger (NHE) gene family J. Exp. Biol., January 11, 2001; 204(21): 3703 - 3716. [Abstract] [Full Text] [PDF]
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 P. Bijlenga, J.-H. Liu, E. Espinos, C.-A. Haenggeli, J. Fischer-Lougheed, C. R. Bader, and L. Bernheim T-type alpha 1H Ca2+ channels are involved in Ca2+ signaling during terminal differentiation (fusion) of human myoblasts PNAS, June 20, 2000; 97(13): 7627 - 7632. [Abstract] [Full Text] [PDF]
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E. Z. Szabo, M. Numata, G. E. Shull, and J. Orlowski Kinetic and Pharmacological Properties of Human Brain Na+/H+ Exchanger Isoform 5 Stably Expressed in Chinese Hamster Ovary Cells J. Biol. Chem., February 25, 2000; 275(9): 6302 - 6307. [Abstract] [Full Text] [PDF]
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 H.-V. Nguyen, G. E Shull, and J. E Melvin Muscarinic receptor-induced acidification in sublingual mucous acinar cells: loss of pH recovery in Na+-H+ exchanger-1 deficient mice J. Physiol., February 15, 2000; 523(1): 139 - 146. [Abstract] [Full Text] [PDF]
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R. L. Evans, S. M. Bell, P. J. Schultheis, G. E. Shull, and J. E. Melvin Targeted Disruption of the Nhe1 Gene Prevents Muscarinic Agonist-induced Up-regulation of Na+/H+ Exchange in Mouse Parotid Acinar Cells J. Biol. Chem., October 8, 1999; 274(41): 29025 - 29030. [Abstract] [Full Text] [PDF]
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N. R. Baird, J. Orlowski, E. Z. Szabo, H. C. Zaun, P. J. Schultheis, A. G. Menon, and G. E. Shull Molecular Cloning, Genomic Organization, and Functional Expression of Na+/H+ Exchanger Isoform 5 (NHE5) from Human Brain J. Biol. Chem., February 12, 1999; 274(7): 4377 - 4382. [Abstract] [Full Text] [PDF]
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