1 Department of Human Physiology, School of Medicine, University of California-Davis, and 2 United States Department of Agriculture-Agricultural Research Service Western Human Nutrition Research Center, Davis, California 95616
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ABSTRACT |
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In this report, we
describe the cloning, cellular localization, and functional
characteristics of Na+/H+ exchanger 1 (NHE1)
from red blood cells of the winter flounder Pseudopleuronectes
americanus (paNHE1). The paNHE1 protein localizes primarily to the
marginal band and exhibits a 74% similarity to the trout -NHE, and
65% to the human NHE1 (hNHE1). Functionally, paNHE1 shares
characteristics of both
-NHE and hNHE1 in that it is activated both
by manipulations that increase cAMP and by cell shrinkage,
respectively. In accordance, the paNHE1 protein exhibits both protein
kinase A consensus sites as in
-NHE and a region of high homology to
that required for shrinkage-dependent activation of hNHE1. After
shrinkage-dependent activation of paNHE1 and resulting activation of a
Cl
/HCO
sodium-proton antiport; red blood cells; -Na+/H+ exchanger; protein kinase A; protein kinase C; protein phosphatases
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INTRODUCTION |
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CELL VOLUME REGULATORY
CAPACITY is a common characteristic of cells ranging from
bacteria and yeast to those of higher mammals, and various mechanisms
of cell volume regulation have been described (24). Red
blood cells (RBCs) have been widely studied as models for cell volume
regulation (10, 12, 14). One early study demonstrated that, after osmotic shrinkage, the RBCs from the winter
flounder, Pseudopleuronectes americanus, exhibited robust NaCl uptake and consequent regulatory volume increase (RVI)
(10). The membrane transport processes and proteins
mediating RVI were, however, not identified. The main membrane
transport mechanisms demonstrated to mediate RVI in most other species
studied are 1) parallel activation of a
Na+/H+ exchanger (NHE) and a
Cl/HCO
cotransport (12,
21, 24). At least six mammalian NHE isoforms (NHE1-NHE6)
with high sequence homology but with varying patterns of distribution
and regulation have been reported (9). NHE1 is the
ubiquitously expressed, "housekeeping" form responsible for
regulation of cell volume and pH and is also the isoform found in
mammalian RBCs (47), whereas the distribution of the other NHE isoforms is more restricted (9, 54). In addition, a
NHE that most closely resembles NHE1 but was termed
-NHE for its potent activation by
-adrenergic agonists has been cloned from hematopoietic tissue from the rainbow trout Oncorhynchus
mykiss (7).
In addition to their activation by osmotic shrinkage, NHE1 from
mammalian species is activated by a wide range of mitogens and growth
factors but is generally inhibited or unaffected by -adrenergic
agonists and other stimuli that increase cAMP (54). A
similar pattern is found in amphibian NHE1s, such as that from Amphiuma RBCs recently cloned by our laboratory
(36), which is activated by osmotic shrinkage
(11) but not by cAMP (P. M. Cala, unpublished
observations). In teleost fish, NHEs differ widely with respect to
activation by osmotic shrinkage and cAMP. The
-NHE is robustly
activated by
-adrenergic stimuli and other manipulations that
increase the cellular level of cAMP (35) but is largely
unaffected by osmotic shrinkage (45, 55). The NHE1
isoforms in RBCs of the brown trout Salmo trutta
(38), the common carp Cyprinus carpio
(39), and the European flounder Platichys
flesus (55) are activated by both cAMP and osmotic shrinkage. Finally, the NHE1 isoform in RBCs of the European eel Anguilla anguilla is most similar to mammalian NHE1 in being
robustly activated by osmotic shrinkage yet unaffected by cAMP
(45, 55). Further complexity is introduced by the fact
that, in some teleost fish, NHE activation by osmotic shrinkage is
inhibited by oxygen, an effect that in European flounder RBCs is so
profound that shrinkage-induced NHE activation is absent at atmospheric
PO2 (8, 55).
Studies employing mutagenesis strongly indicate that direct
phosphorylation by protein kinase A (PKA) plays an important role in
the activation of -NHE by cAMP (6). With respect to
shrinkage-induced NHE1 activation, however, the role of protein
phosphorylation is more controversial, and the precise relationship
between phosphorylation and NHE1 activity is incompletely understood.
Thus, although several studies (16, 43, 51, 54) strongly
suggest the involvement of serine/threonine protein phosphorylation in
shrinkage-induced NHE1 activation, there is no evidence that this
reflects direct phosphorylation of the NHE1 protein (4,
18). Further evidence that NHE1 activity can be induced by
protein phosphorylation comes from studies demonstrating the activation
or stimulation of NHE1 by exposure to okadaic acid or calyculin A
(CL-A), inhibitors of serine/threonine protein phosphatase (PP) 1 and
PP2A (5, 19, 43, 48). The mechanism by which PP inhibitors
activate NHE1 is not elucidated. A role for direct NHE1 phosphorylation in okadaic acid-mediated activation was suggested in mammalian cells
(5, 48). Activation of
-NHE by CL-A was proposed to involve recruitment of an intracellular pool of exchangers to the
plasma membrane (19), and a similar mechanism may be
involved in CL-A-mediated NHE1 activation in Amphiuma RBCs
(A. Ortiz-Acevedo, R. R. Rigor, and P.M. Cala, unpublished
observations). Finally, the possible relationship between NHE
activation by cAMP, shrinkage, and phosphatase inhibitors has so far
not been studied in any species.
Given the robust RVI seen in winter flounder RBCs kept at atmospheric PO2 (10) and the close relationship of this species to the European flounder in which NHE1 is cAMP-activated, we hypothesized that the winter flounder RBCs might possess a NHE activated by cAMP, osmotic shrinkage, and phosphatase inhibitors at atmospheric PO2, making it a valuable tool to study the involvement and possible interrelationship of these stimuli in NHE activation.
The aim of the present study, therefore, was to determine whether a NHE mediates the shrinkage-activated Na+ influx pathway in the RBCs of the winter flounder P. americanus and to investigate the regulation of this pathway by osmotic shrinkage, increases in cellular cAMP levels, and general increases in serine/threonine protein phosphorylation. To do this, we have cloned and sequenced the NHE1 from winter flounder RBCs, studied the presence and subcellular localization of the NHE1 protein in these cells, and analyzed its activation by osmotic shrinkage, cAMP, and the serine/threonine phosphatase inhibitor CL-A, separately and in combination.
Part of these results have previously been published in abstract form (26, 27, 41, 42).
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MATERIALS AND METHODS |
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Materials, Stock Solutions, and Experimental Media
Unless otherwise stated, chemicals were of molecular biology or analytical grade and were obtained from Fisher Scientific (Houston, TX) or Sigma Aldrich (St. Louis, MO).Stock solutions of isoproterenol (Calbiochem, La Jolla, CA) were
prepared in distilled H2O, and stocks of H89, Gö
6850, and CL-A (also Calbiochem) were prepared in DMSO. All these
stocks were stored at 20°C until use. Stock solutions of
4,4'-diisothicyanate-2,2'-stilbene-disulfonic acid (DIDS) were prepared
in the isotonic Ringer immediately before use. Ouabain was dissolved
directly in the media at a concentration of 1 mM. The standard isotonic
HEPES Ringer had an osmolarity of 360 mosM and contained (in mM) 148 NaCl, 3 KCl, 1 MgCl2, 0.75 CaCl2, 30 HEPES,
adjusted to pH 7.65 with NaOH. Hypertonic solutions were made by
increasing the concentration of NaCl. The Na+-free medium
was made by substituting N-methyl-D-glucamine
(NMDG)-Cl and NMDG-OH for, respectively, NaCl and NaOH. In the poorly
buffered media, the concentration of HEPES was reduced to 0.5 mM and
that of NaCl increased correspondingly to maintain osmolarity;
otherwise, ionic composition was unaltered compared with the standard
media. Experimental media were gassed with air saturated with
H2O for about 5 min before use.
Animals and RBC Preparation
Adult specimens of winter flounder (P. americanus) were maintained in seawater at 10°C. RBCs were prepared essentially as previously described (10). Briefly, blood was drawn by caudal vein puncture with a heparinized syringe, followed by mild centrifugation (60 s, 1,800 g), removal of plasma and buffy coat by aspiration, and resuspension in 8-10 volumes of the standard isotonic medium. After two additional washes in isotonic medium, cells were resuspended at 10% hematocrit in this medium and preincubated for 2 h in the dark at room temperature before the start of experiments. All experiments were conducted at room temperature (20-25°C).The winter flounder plasma had a mean osmolarity of 323 ± 1.2 mosM (n = 13 fish) and a pH of 7.62 ± 0.025 (n = 13 fish).
RNA Isolation, Reverse Transcription, and cDNA Amplification
Total RNA (SV Total RNA isolation system, Promega) and mRNA (PolyATract System 1000, Promega) was extracted from winter flounder RBCs by using standard methods. Total RNA or mRNA was reverse transcribed by using a mix of random hexameric and oligo dT primers (both Invitrogen). The cloning strategy and primers used to amplify the winter flounder NHE1 are illustrated in Fig. 1. An initial ~900-bp fragment of NHE1 was amplified from cDNA by using primers designed on the basis of the previously published sequences for Amphiuma tridactylum NHE1 (36), human NHE1 (49), and trout
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Sequencing and Sequence Analysis
PCR products were gel purified, sequenced (dideoxy chain termination method, the DBS Sequencing Facility, University of California-Davis), and analyzed for homologies to known nucleic acid sequences using the blastn program (1). Alignments of NHE nucleotide and amino acid sequences were performed by using Genetics Computer Group (GCG) software and the BLOSUM62 matrix. Construction of phylogenetic trees was also performed in GCG software, using Kimura protein distance correction and with distance calculated from the estimated number of substitutions per 100 amino acids. Hydropathy analysis (31) was performed by using the TMpred, PSORT II, and GCG programs. Protein kinase consensus sequences were detected by using the PROSITE database (2).Immunoblotting and Immunocytochemistry
Mouse monoclonal antibody 4E9 (kindly provided by Dr. D. Biemesderfer, Yale University, School of Medicine) was produced against a fusion protein containing the cytoplasmic region of porcine NHE1 (amino acids 514-818). 4E9 has been shown to specifically bind NHE1, but not other NHE isoformsImmunoblotting. Crude membrane preparations were made by lysis of 0.5 ml of packed RBCs in 10 ml of water containing a cocktail of protease inhibitors. The membrane pellet after a 60-min, 150,000 g centrifugation was dissolved in sample buffer, resolved by 7.5% SDS-PAGE, and transferred onto PVDF membrane. The membrane was blocked for 60 min at room temperature (5% non-fat dry milk in PBST), incubated for 1 h with primary antibody (4E9 at 1:10,000, XB-17 at 1:1,000 or polyclonal antibody 666 at 1:3,000), washed (PBST, 5 min × 3), incubated in horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h, washed (PBST 5 min × 5, PBS 15 min × 1), and visualized by chemiluminescence.
Immunocytochemistry. RBCs were washed three times in isotonic medium and incubated at 10% hematocrit overnight at 4°C. A 0.1-ml aliquot of packed cells was fixed by incubation in 4 ml of 3% paraformaldehyde in PBS overnight at 4°C, and fresh 3% paraformaldehyde solution was substituted the next day. One milliliter of the cell suspension was transferred into a 1.5-ml centrifuge tube. Cells were washed twice in PBS containing 0.5% Triton X-100 and permeabilized in the same medium for 15 min at room temperature. The permeabilized cells were washed twice with blocking buffer (3% BSA in PBS-0.5% Triton X-100) and incubated in the blocking buffer for 1 h, followed by incubation with primary antibody (XB-17 at 1:100 in the blocking buffer) for 1 h at room temperature. Cells were washed three times in PBS and incubated with FITC-conjugated goat anti-rabbit IgG at 1:200 in the blocking buffer for 1 h, followed by extensive washing in PBS-0.5% Triton X-100 and a final wash in PBS and Cells were resuspended in 0.5 ml of PBS, and a 0.1-ml aliquot was transferred onto a no. 1 cover slip. Cells were observed with a 63×/1.4 NA oil DIC objective mounted on an inverted microscope (LSM 510 confocal system, ZEISS), using an excitation wavelength of 488 nm (Argon laser, 20% power), emission filter band path of 505-550, and a pinhole of 2 airy disc size. A projected image was produced with a stack of Z sections (7.2 µm) of a representative image area.
Net Flux Measurements
Determination of cellular content of Na+, K+, ClMeasurements of Extracellular pH in Poorly Buffered Media
Cells were preincubated with or without 10 µM DIDS for 2 × 20 min in the standard isotonic HEPES ringer, followed by two washes in poorly buffered isotonic medium containing 1 mM ouabain and resuspension in this medium at 10% hematocrit. Seven hundred microliter aliquots were pelleted by brief centrifugation and resuspended in ouabain-containing iso- or hypertonic poorly buffered medium at time 0. Extracellular pH (pHo) was monitored by using pH combination microelectrodes (Microelectrodes, Bedford, NH) coupled to a Powerlab/400 and Chart 4.1.1 software (ADInstruments, Grand Junction, CO). ![]() |
RESULTS |
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cDNA and Nucleic Acid Sequences of the NHE from Winter Flounder RBCs
The cloning strategy employed is illustrated in Fig. 1. An initial ~0.9-kb fragment was amplified from cDNA from winter flounder RBCs by using primers based on the cDNA sequences of human and Amphiuma NHE1s and trout
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Hydropathy analysis of the winter flounder NHE in TMpred using the Kyte
and Doolittle algorithm (31) and allowing transmembrane (TM) regions of 17-33 amino acids predicts 12 TM regions. A
hydropathy analysis in PSORT II, using an newer algorithm modified from
(28), which allows for the fact that less hydrophobic
amino acids are more easily integrated into the membrane once a part of
the peptide is integrated, also predicts 12 TM regions. However, two
regions with relatively low significance scores differ in the two
predictions, and closer analysis of the hydropathy scores reveals that
there are in fact 13 potential, membrane-embedded regions,
corresponding well with the 12 TM regions plus 1 hydrophobic region
"dipping" into the membrane from the extracellular side recently
described for the human NHE1 (53). Furthermore, PSORT II
prediction using an algorithm by Hartmann et al. (23)
indicates that both the NH2 and COOH termini are
intracellular. Taken together, these data indicate that the flounder
NHE most probably consists of a short cytoplasmic
NH2 terminus, an amphipathic region with 12 TM domains and
a hydrophobic region between TM9 and TM10 that dips into the membrane
from the extracellular side, and a COOH-terminal hydrophilic tail
beginning at amino acid 489. The probable TM regions and the dip are
underlined in Fig.
3B.
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Figure 3A shows a cladogram illustrating the resemblance of
the flounder RBC protein to other cloned NHEs. Compared with the human
NHE isoforms, the flounder RBC NHE is most closely related to NHE1,
with 65% identity/71% similarity at the amino acid level, 46%/54%
with NHE2, and 41%/51% with NHE3 (all identity and similarity data
were calculated in GCG software using the BLOSUM62 amino acid
substitution matrix). We will therefore refer to it as
Pseudopleuronectes americances (pa)NHE1. Compared with other
NHE1s, the paNHE1 protein exhibits the highest homology with NHE1 from
the European flounder (95% identity/96% similarity), followed by
trout -NHE (74%/79%), common carp NHE1 (72%/77%),
Amphiuma giant salamander NHE1 (65%/71%), and European eel
NHE1 (57%/63%). Similar homologies were found at the nucleic
acid level (not shown). The homology of paNHE1 with the other cloned
NHEs is highest in the NH2-terminal region (amino acids
1-489). Thus this region exhibits 85%/89% and 78%/83% identity/similarity with
-NHE and human NHE1, respectively, compared with 66%/75% and 56%/65%, respectively, in the COOH-terminal tail region (amino acids 490-779). The very NH2-terminal
end of the NHE1s (approximately amino acids 1-50) shows minimal
homology between the NHE1s and has been suggested to be a cleavable
signal peptide, although this has been disputed by some recent findings (53). Database searching using PSORT II based on the von
Heijne algorithm (52) does not suggest the presence of an
NH2-terminal signal peptide in paNHE1, although the
sequence does contain a putative cleavage site between amino acid 40 and 41.
Figure 3B shows an alignment of the paNHE1 amino acid
sequence with those of a mammalian NHE1 (human), an amphibian
(Amphiuma) NHE1, and trout -NHE; the paNHE1 motifs
described below are marked in the figure. The paNHE1 exhibits four
putative N-glycosylation sites (N50, N72, N361, and N400),
of which N72 corresponds to N75 in the human NHE1, which is
N-glycosylated in vivo. In contrast to the human NHE1,
paNHE1 does not contain putative O-glycosylation sites. The
paNHE1 also contains two putative PKA/protein kinase G (PKG)
phosphorylation sites, KKES (S503) and RRMS (S669), both of which are
shared by
-NHE (corresponding to S481 and S648, respectively, in the
-NHE protein). In addition, the hydrophobic tail region contains
three putative protein kinase C (PKC) phosphorylation sites (S503,
S704, and T727).
Presence and Subcellular Localization of the paNHE1 Protein in Winter Flounder RBCs
Figure 4A shows Western blots of winter flounder RBC membrane fractions with 4E9, a monoclonal antibody against NHE1. As seen, the antibody recognizes a band of ~100 kDa. This is slightly higher than the calculated molecular weight of paNHE1, as expected for a glycosylated protein, and in good agreement with findings for NHE1 from other species (9, 54). The 4E9 antibody is raised against a fusion protein containing amino acids 514-818 of the porcine NHE1 and was found not to label
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Confocal imaging of fixed, permeabilized winter flounder RBCs labeled with a polyclonal NHE1 antibody (XB-17) revealed that paNHE1 is primarily localized to the marginal band of the RBCs (Fig. 4B). Fainter, at least partly punctate intracellular labeling was also detectable in the labeled cells. Only very faint, diffuse labeling could be seen in control cells labeled with secondary antibody only. Lending further credence to the conclusion that the labeling pattern in flounder RBCs reflects the distribution of paNHE1, it may be noted that in chicken and Peking duck RBCs, which lack volume-regulatory NHE activity (P. M. Cala, unpublished observations; Ref. 34), there was no detectable labeling with XB-17 in either confocal images or Western blots (not shown, n = 2 of each for chicken, n = 1 of each for duck).
Effect of Osmotic Shrinkage on paNHE1, and Mechanism of RVI in Winter Flounder RBCs
Cala (10) demonstrated robust, shrinkage-induced NaCl uptake and RVI by yet unidentified mechanisms in winter flounder RBCs at atmospheric O2 pressure. This is in contrast to findings in RBCs of the closely related European flounder, where shrinkage-induced Na+ influx was negligible except at very low PO2 (55). We therefore pursued this question, initially investigating the functional identity of the shrinkage-activated transporters responsible for RVI in winter flounder RBCs. All experiments in the present study were performed at atmospheric PO2.As noted in the introduction, two types of membrane transport
mechanisms mediate ion uptake during RVI in most cells. One involves
shrinkage-induced activation of a NHE leading to an increase in
intracellular pH (pHi), which entrains net transport
through a Cl/HCO
cotransport (12,
24). Figure 5 shows the results of
experiments carried out to investigate the molecular mechanism of RVI
in winter flounder RBCs. As previously shown (10),
exposure to hypertonic medium in the presence of 1 mM ouabain (to
inhibit the Na+-K+-ATPase) elicits an increase
in intracellular Na+ (Fig. 5A) and
Cl
(Fig. 5B) content, with little change in
intracellular K+ content (not shown). When cells were
pretreated with DIDS (10 µM) to inhibit the
Cl
/HCO
content is abolished (Fig. 5B), whereas the
increase in Na+ content is unaffected (Fig. 5A).
Figure 5C illustrates experiments in which pHo
was monitored over time in suspensions of cells in poorly buffered iso-
or hypertonic media. As shown, in control cell suspensions, the change
in pHo over time is minimal and similar in iso- and
hypertonic media. In contrast, in DIDS-pretreated cells, osmotic
shrinkage elicits a significant decrease in pHo, whereas
the pHo of isotonic cell suspensions is only marginally affected. Taken together, these data strongly suggest that the ion and
water fluxes induced by osmotic shrinkage of winter flounder RBCs are
mediated by the parallel activity of a NHE and a
Cl
/HCO
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The effect of the magnitude of hypertonic challenge on intracellular
ion and water content in the winter flounder RBCs is illustrated in
Fig. 6. The degree of cell shrinkage,
illustrated by the decrease in cellular H2O content (Fig.
6A), increases with increasing hypertonic challenge, with
cell shrinkage detectable at the mildest hypertonic challenge tested
(1.1 times isotonic osmolarity). Despite this, increases in
intracellular Na+ (Fig. 6B) and Cl
(Fig. 6C) content are only seen after hypertonic challenges
of 1.5 times isotonic or above, indicating the existence of a
"threshold" for activation of the transporters. As illustrated in
Fig. 6B, the Na+ uptake is proportional to the
medium osmolarity and therefore to the osmotic cell shrinkage. The
K+ content, as expected, is marginally affected by
hypertonic challenges in the range tested (Fig. 6D). It is
noteworthy that the measured H2O gain during RVI is less
than that expected on the basis of the measured changes in cellular
Na+, K+, and Cl
content (see
DISCUSSION).
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Activation of paNHE1 by Increases in the Cellular cAMP Level
The changes in cellular ion and water content as a function of time after stimulation with the
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Activation of paNHE1 by the Serine/Threonine Phosphatase Inhibitor CL-A
Figure 9 illustrates the changes in intracellular ion and H2O content as a function of time after stimulation with 100 nM of the serine/threonine phosphatase inhibitor, CL-A. CL-A treatment elicits robust increases in the cellular Na+, Cl
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Additivity of paNHE1 Activation by Osmotic Shrinkage, cAMP, and CL-A
To address the possible interrelationship between paNHE1 activation by osmotic shrinkage, increases in cAMP, and CL-A, we assessed the possible additivity of the effects of these stimuli. As seen in Fig. 11, stimulation with a maximal dose of isoproterenol (10 µM) given simultaneously with a maximal dose of CL-A (100 nM) elicited increases in cellular Na+ and H2O content that were at least as large as the sum of the two individual stimuli. Similarly, a maximal stimulation with either isoproterenol (10 µM) or CL-A (100 nM) was at least additive to activation by hypertonic challenge (1.5 times isotonic) (Fig. 11, C and D). Similar findings were obtained for cellular Cl
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DISCUSSION |
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Mammalian NHE1 is activated by a range of stimuli, including osmotic cell shrinkage, but are generally inhibited or unaffected by stimuli that increase cAMP (see Ref. 54). In contrast, NHE1s of teleost fish are generally (although not universally; see Ref. 45) activated by increases in cAMP, whereas the effect of osmotic shrinkage ranges from essentially absent to robust and is often strongly PO2 dependent (14, 35, 38, 39, 55). The signal transduction mechanisms involved in NHE1 activation, and the role of protein phosphorylation in particular, remain incompletely understood (for a discussion, see Ref. 54). The present paper describes the cloning, sequencing, and localization pattern of NHE1 from winter flounder RBCs, demonstrates its activation by osmotic shrinkage, by increases in cellular cAMP levels, and by the serine/threonine phosphatase inhibitor CL-A, and addresses the question of whether these stimuli activate NHE1 by distinct or shared signaling mechanisms.
NHE1 from Winter Flounder RBCs: Transcripts, and Protein Characteristics and Localization
The NHE cDNA amplified from the flounder RBCs was found to encode a protein of 779 amino acids with a calculated molecular mass of 86.6 kDa. This protein most closely resembles NHE1 and has hence been termed paNHE1. It may be noted that three partially different 3' UTRs were found by 3' RACE, whereas the 5' UTR and ORF were identical with the exception of three silent dimorphisms. Because teleost RBCs can remain in the circulation for up to 6 mo (see Ref. 33), this may reflect the well-described phenomenon of developmental differences in 3' UTR sequence (17) and/or the existence of several paNHE1 gene copies. Given the extensive washing of the blood and removal of any detectable non-red cell contaminants, as well as the 100% identity of the sequences complementary to both the forward and reverse 3' RACE primers, it seems less likely that the consistent detection of several clones reflects contributions to the preparation from non-red cells, although this possibility cannot be excluded. For the purpose of the present study, however, this was not further investigated. In other flounder tissues (gill, small intestine, kidney and heart), a transcript of ~7.5 kb was detected by Northern blotting with a probe encoding the membrane-spanning region of human NHE1 (22). Among the NHE1s from various species, paNHE1 exhibits the highest homology, with NHE1 from the European flounder Platichtys flesus (Genbank accession number AJ006918) followed by rainbow troutNature of the Shrinkage-Activated Ion Flux Pathways and Activation of paNHE1 by Multiple Stimuli
It has long been known that winter flounder RBCs exhibit RVI by uptake of NaCl and osmotically obliged water with no increase in cellular K+ content (10). In the present study, we confirmed these findings, and further showed that the increase in ClBecause of the low water-to-solute ratio in the hyperosmotic media used
in the RVI experiments (see also below), the H2O gain during RVI was modest. In addition, the measured H2O gain
during RVI was generally somewhat smaller than that expected on the
basis of the changes in cellular Na+, K+, and
Cl content. Our present speculation is that the uptake of
inorganic ions is, at least in part, offset by a loss of organic ions;
however, this remains to be investigated. This phenomenon was only seen during RVI, whereas after stimulation with isoproterenol or CL-A the
measured and calculated H2O gain were identical.
In contrast to the findings by Cala (10), the RVI after
direct hyperosmotic shrinkage as employed in the present study was not
complete after 90 min. This is a common phenomenon after direct hyperosmotic shrinkage in many cell types, whereas the so-called regulatory volume decrease (RVD)-RVI protocol of osmotic shrinkage employed by Cala (10) leads to more complete volume
recovery. Briefly, in the RVD-RVI protocol, cells are swollen in
hypotonic media, allowed to regulate volume, and subsequently
transferred to the original, control isosmotic medium. The control
medium is now hypertonic because of volume regulatory solute loss in hyposmotic medium. Because the medium in which cells are shrunken in
the RVD-RVI protocol is hypertonic yet not truly hyperosmotic, the
volume of osmotically obliged water associated with each millimole of
solute transported during RVI is greater than for cells shrunken by
direct transfer from isosmotic to hyperosmotic medium. Thus we believe
that the difference between the present data and that of Ref.
10 is at least in part a reflection of the lower
osmolarity of the solution in which cells are shrunken and volume is
regulated when the RVD-RVI protocol is employed. Although this explains the difference in water uptake, it does not address the basis for
limitation of Na+ uptake during RVI, which is generally due
to the fact that pHi rises above the pHi set
point, resulting in NHE1 deactivation, i.e., the deactivating effect of
elevated pHi overrides the activating effect of reduced
cell volume. However, in the case of paNHE1, we believe that incomplete
volume regulation during RVI in the present study is a reflection of
slow time course rather than pH-dependent deactivation of
shrinkage-induced Na flux or dissipated driving force for the following
reasons: 1) at all osmolarities tested, the driving force
for inward Na+ flux via NHE1
(µNa+
µH+) was substantial throughout the 90-min time course of the
experiment; 2) the pHi calculated from the
chloride distribution ratio
([H+]i/[H+]o = [Cl
]o/[Cl
]i)
increased in proportion to Na+ flux in all hypertonic media
and was proportional to medium osmolarity. If volume regulation were
curtailed by paNHE1 deactivation dictated by pHi set point,
then the pH at which flux begins to decline should be the same in all
cases. However, at 90 min, total Na+ uptake at 2.0 × isotonic osmolarity was nearly twice that in 1.75 × isotonic
osmolarity, although pHi was similar, ~7.40, in both
conditions (the calculated pHi of unstimulated
cells in isotonic medium is
~7.25-7.30).1 Although
further studies are needed to directly address this issue, this could
indicate that when shrinkage activates, paNHE1 is not turned off by an
increase in pHi. Consistent with this notion is the
observation that Na+ uptake during RVI is not significantly
inhibited in DIDS-treated cells (Fig. 5), where pHI is
expected to increase to >7.45 because the
Cl
/HCO
Stimulation of the RBCs with the -adrenergic agonist isoproterenol
or direct activation of the adenylate cyclase by forskolin also
elicited rapid and robust increases in the cellular content of
Na+, Cl
, and H2O, but not
K+, consistent with activation of paNHE1 by increases in
the cellular level of cAMP. Finally, exposure to the serine/threonine
phosphatase inhibitor CL-A elicited dose-dependent increases in
Na+, Cl
, and H2O content in the
flounder RBCs, which were similar in magnitude but slower in time
course than those elicited by isoproterenol. Cellular K+
content was essentially unaltered by CL-A treatment. Similar to what
was found for RVI, the pHi calculated from the chloride distribution ratio increased in proportion to the increase in Na+ after both isoproterenol and CL-A treatment (not
shown). The ion and water net flux data thus strongly indicate that the
Na+ uptake after isoproterenol or CL-A treatment, as well
as after osmotic shrinkage, reflects the activity of a NHE.
Furthermore, given, that the paNHE1 sequence is consistent with
activation by both osmotic shrinkage, increases in cAMP, and inhibition
of protein phosphatases (see below), and that Western blotting and PCR
failed to detect the presence of other NHE isoforms, it seems highly
probably that paNHE1 is the transporter mediating the Na+
uptake in response to all three stimuli.
Mechanisms of paNHE1 Activation: Comparison with NHE1s from Other Species
Shrinkage-induced activation of human NHE1 was inhibited 80% by deletion of the high-affinity calmodulin-binding domain (amino acids 636-656) (3), and ablated in aUnder the experimental conditions employed in this study, shrinkage-mediated activation of paNHE1 was fully additive to activation by CL-A. The fact that CL-A activates paNHE1 under isotonic conditions indicates that a serine/threonine protein kinase(s) involved (directly or indirectly) in activation of paNHE1 is at least partly active under these conditions, as is the corresponding phosphatase(s). Further studies are required to determine whether shrinkage leads to activation of paNHE1 by activation of a protein kinase, as indicated by studies on mammalian RBCs (25, 40) or by inhibition of a (CL-A-sensitive) protein phosphatase. Future studies will also address the identity of the putative shrinkage-activated kinase, but it may be noted that direct phosphorylation by a tyrosine kinase can be ruled out by the lack of consensus sites for tyrosine phosphorylation and that preliminary findings using the PKC inhibitor Gö 6850 argue against a role for PKC in shrinkage-induced paNHE1 activation (S. A. King, S. F. Pedersen, R. Rigor, Z. Zhuang, and P. M. Cala, unpublished observations).
In some (37), although not all (30), cells
studied, osmotic shrinkage is associated with an increase in cAMP,
potentially leading to PKA-mediated NHE1 activation. However, in the
present study, stimulation of paNHE1 by shrinkage was at least additive to a maximal stimulation by cAMP, indicating that these stimuli activate paNHE1 through distinct mechanisms. The mechanism of cAMP-mediated NHE1 activation has only been studied in detail for
-NHE, in which glycine substitution of two PKA consensus sites, S641
and S648, reduced cAMP-mediated activation by 70% (6).
The paNHE1 protein has two putative PKA/PKG phosphorylation sites, KKES
(S503) and RRMS (S669). S669 is the most likely to be PKA
phosphorylated in vivo (50) and corresponds to S648 in
-NHE. S503 is also shared by
-NHE, but the corresponding site,
NHE S481, does not appear to contribute to the cAMP-mediated activation of
-NHE, because this activation is ablated in a
559 mutant (6). In support of the notion that PKA-mediated
phosphorylation of this motif plays a major role, the S648 motif of the
-NHE is shared by all of the cAMP-activated NHE1s, whereas it is
absent in the eel, mammalian, and amphibian NHE1, none of which are
significantly activated by cAMP (Refs. 45,
54; P. M. Cala unpublished observation for
Amphiuma NHE1). PKA-independent effectors of cAMP (e.g.,
Ref. 32 and references therein) could, however, also
contribute to cAMP-mediated NHE1 activation.
CL-A- and Isoproterenol-induced Cell Swelling: Lack of Robust K+ Loss and RVD
In marked contrast to findings in Amphiuma RBCs (A. Ortiz-Acevedo, H. M. Maldonado, R. Rigor, and P. M. Cala, unpublished observations), but in agreement with findings in trout (19) and eel (45) RBCs, CL-A treatment elicited only very modest K+ loss from winter flounder RBCs. The little K+ loss that did occur probably reflects swelling-induced activation of a K+ efflux pathway, which in these cells appears to be primarily KCl cotransport (24). Although not further studied here, the absence of robust compensatory K+ loss and RVD is conspicuous, given the massive cell swelling induced by CL-A treatment. A slightly larger K+ loss was seen at late time points after isoproterenol stimulation, but analogously to what was seen after CL-A-treatment, the K+ efflux was insufficient to elicit any RVD in the time period studied. An apparently similar phenomenon was described in duck RBCs, in which noradrenaline treatment prevented swelling-induced activation of KCl cotransport (20). Even the massive swelling induced by the combined stimulation with CL-A and isoproterenol elicited only very slight compensatory K+ efflux, and RVD was completely absent (n = 3, data not shown). This, interestingly, is consistent with the notion (25, 40) that a swelling-induced increase in the activity of a protein phosphatase relative to that of a protein kinase is required for swelling-induced activation of net K+ loss.Finally, the cAMP-mediated paNHE1 activation was fully additive to
CL-A-mediated activation. A similar phenomenon was also observed in
trout RBCs, where it was tentatively proposed that the two stimuli act
on different -NHE pools, with CL-A recruiting otherwise silent
exchangers from intracellular pools (19). In mammalian
cells (5, 48), activation of NHE1 by okadaic acid was
associated with an increase in direct phosphorylation of NHE1. The
present findings are compatible with several possible models that
should be tested in future studies. CL-A could act not by a separate
pathway per se but rather by shifting the kinase-phosphatase equilibria
involved in shrinkage- and cAMP-mediated activation in the direction of
increased phosphorylation. Because all of the relevant regulatory
molecules should eventually be shifted to the phosphorylated form in
the presence of high concentrations of CL-A, this scenario is only
likely in the relatively short term and only if the tonic activity of
the corresponding kinase is sufficiently low that an increase in kinase
activity causes a distinctly more rapid shift toward the phosphorylated
state in the presence of CL-A. Alternatively, paNHE1 could exhibit
several activated states, induced by osmotic shrinkage, cAMP, and CL-A, each by distinct mechanisms. The effect of CL-A could be on exchangers resident in the plasma membrane or could involve increased recruitment from intracellular pools as suggested for
-NHE. In the latter case,
however, such intracellular pools of paNHE1 would have to be located
close to the plasma membrane, since we found the paNHE1 protein to be
primarily located in or very close to the plasma membrane.
Physiological and Phylogenetic Considerations
The ability to regulate cell volume is a common feature of most cells and one of fundamental importance for the cells of euryhaline, migratory fish. In euryhaline fishes going from freshwater to seawater, significant RBC shrinkage has been observed (8); hence, RVI by activation of NHE1 would seem to be of high adaptive significance. Paradoxically, the degree of sensitivity of NHE1 to osmotic shrinkage differs greatly between teleost species in a manner apparently unrelated to euryhalinity. Thus the winter flounder (this study) and carp, which are only mildly euryhaline (brackish water to seawater), and the eel, which is highly euryhaline given its migration between freshwater and seawater, all exhibit robust shrinkage-induced NHE1 activation, whereasActivation of NHE1 by a cAMP-signaling pathway is thought to be of
importance in fish during plasma hypoxia and acidification (e.g., Ref.
39). In addition to the decrease in pHi
associated with these conditions, which will in itself activate NHE1,
the increased plasma concentrations of catecholamines activate
adrenergic receptors. The -adrenergic receptor activates adenylate
cyclase, increasing the cellular level of cAMP, thus activating PKA.
Activation of NHE1, presumably via PKA, leads to increased
H+ efflux, which in turn leads to increased oxygen
saturation of hemoglobin at any given PO2 (the
Bohr effect). The swelling that occurs secondary to NHE1 activation
will also contribute to increased oxygen saturation by diluting the
cellular concentrations of hemoglobin and phosphate (8).
In some teleosts, NHE1 is strongly inhibited by oxygen, by a mechanism
that is not understood but has been proposed to involve hemoglobin,
possibly bound to AE1, as an oxygen sensor (15). Under
conditions of hypoxia, an increase in NHE1 activity can thus be induced
both by disinhibition as a consequence of the decrease in
PO2 and by direct activation by the
-adrenergically mediated increase in cAMP. Oxygen sensitivity
differs greatly between teleost species (15), and it is
interesting to note that paNHE1 exhibits substantial activation by
osmotic shrinkage in an air atmosphere (this study and Ref.
10), whereas the NHE1 from European flounder RBCs is only
activated by osmotic shrinkage at low PO2
(55). Given the high sequence homology of these NHE1s (95% identity at the amino acid level), this suggests that the difference in oxygen sensitivity is to be found at the level of other
cellular components rather than at the level of NHE1 itself. Moreover,
the at-least-additive effect of shrinkage and isoproterenol seen in the
present study was not observed for the European flounder NHE1
(55). Because the European flounder studies were conducted at low PO2, it is possible that this difference
may relate to the difference in PO2 in the two studies.
In conclusion, the present report describes the cloning, cellular
localization, and functional characteristics of NHE1 (paNHE1) from RBCs
of the winter flounder P. americanus. The paNHE1 protein localizes primarily to the marginal band and exhibits 74% similarity to trout -NHE and 65% to the human NHE1. Functionally, paNHE1 shares characteristics of both
-NHE and human NHE1 in that it is
activated both by manipulations that increase cAMP and by cell shrinkage, respectively. In accordance, the paNHE1 protein exhibits both PKA consensus sites, as in
-NHE, and a region of high homology to that required for shrinkage-dependent activation of hNHE1. After
shrinkage-dependent activation of paNHE1 and resulting activation of a
Cl
/HCO
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ACKNOWLEDGEMENTS |
---|
We are grateful to W. Goschberg and S. Sligh for technical assistance, and to the Burroughs Wellcome foundation for financial support for these technicians. We also thank Dr. L. Renfro, University of Connecticut, Storrs, CT, for help in obtaining the winter flounder RBCs, and Dr. D. Biemesderfer (Yale University, New Haven, CT), Dr. M. Musch (University of Chicago, Chicago, IL), and Dr. J.B. Claiborne (Georgia Southern University) for kindly providing us with, respectively, the 4E9, XB-17, and ab 666 antibodies.
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FOOTNOTES |
---|
The present study was supported by the National Heart, Lung, and Blood Institute Grant HL-21179 (to P. M. Cala), the Bodil Schmidt-Nielsen fund (to P. M. Cala), Carlsberg Foundation Grant 0544/20 (to S. F. Pedersen), and the Philip Morris Foundation (to S. A. King).
The paNHE1 sequence has been submitted to GenBank (accession number AY167041).
Address for reprint requests and other correspondence: S. F. Pedersen, Dept. of Human Physiology, School of Medicine, Tupper Hall, Univ. of California, 1 Shields Ave., Davis, CA 95616 (E-mail: sfpedersen{at}aki.ku.dk).
1
It may be noted that the pHi
similarity in the two conditions reflects that net ion flux via the
Cl/HCO
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.
First published January 29, 2003;10.1152/ajpcell.00562.2002
Received 3 December 2002; accepted in final form 27 January 2003.
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