Molecular cloning of NHE1 from winter flounder RBCs: activation by osmotic shrinkage, cAMP, and calyculin A

Stine F. Pedersen1, Scott A. King1, Robert R. Rigor1, Zhenpeng Zhuang1, Jaimie M. Warren2, and Peter M. Cala1

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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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 beta -NHE, and 65% to the human NHE1 (hNHE1). Functionally, paNHE1 shares characteristics of both beta -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 beta -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<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger, their parallel operation results in net uptake of NaCl and osmotically obliged water. Activation of paNHE1 by cAMP is at least additive to that elicited by osmotic shrinkage, suggesting that these stimuli regulate paNHE1 by distinct mechanisms. Finally, exposure to the serine/threonine phosphatase inhibitor calyculin A potently activates paNHE1, and this activation is also additive to that induced by shrinkage or cAMP.

sodium-proton antiport; red blood cells; beta -Na+/H+ exchanger; protein kinase A; protein kinase C; protein phosphatases


    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<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger, as was first demonstrated in our laboratory (11) in 1980 in studies on RBCs of the giant salamander Amphiuma tridactylum; or 2) Na+-K+-2Cl- 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 beta -NHE for its potent activation by beta -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 beta -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 beta -NHE is robustly activated by beta -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 beta -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 beta -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, 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 beta -NHE (7) (primer pair 1). An additional ~1,000 bp of sequence upstream to this fragment was then obtained by using primer pair 2, whose design was based on the fragment obtained with primer pair 1 and the sequence of the European flounder NHE1 (Genbank accession no. AJ006918). The remaining upstream fragment of the open reading frame (ORF) was obtained by using primer pair 3 (designed from the fragment obtained with primer pair 2 and the start of the trout beta -NHE1 ORF). A band of the expected size, amplified with primer pair 3, was then extracted from the gel and reamplified by using an abbreviated sequence of the forward primer 3 and a portion of the reverse primer 3 that extended an additional two nucleotides into the predicted NHE1 sequence (primer pair 4). Sequences of the 5' and 3' untranslated regions (UTRs) were obtained by using the rapid amplification of cDNA ends procedure (RACE) (SMART RACE cDNA amplification kit, Clontech), with primers specific for regions in the winter flounder NHE1 ORF. The protocol used for standard PCR reactions were 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 45-55°C (optimized to the melting temperature of each primer pair used) for 45 s, and 72°C for 2 min. This was followed by a single cycle of 72°C for 10 min. RACE reactions were carried out as follows: 5 cycles of 94°C for 5 s and 72°C for 3 min; 5 cycles 94°C for 5 s, 69°C for 10 s, and 72°C for 3 min; 25 cycles of 94°C for 5 s, 67°C for 10 s, and 72°C for 3 min. The full ORF was then amplified by using primers with Hind III and Xho I restriction enzyme sites integrated in the 5' ends of the forward and reverse primers, respectively, i.e., 5'-TTGGTAAAGCTTCTGAAAATGGCTGCTCTCTTGCTCCG-3' (forward with Hind III) and 5'-TATATACTCGAGATTCCTCATGAGACGAAGGAGCCGTCCT-3' (reverse with Xho I), cloned into the pcDNA3.1(+) vector (Invitrogen), and sequenced for verification. The full length P. americanus NHE1 (paNHE1) cDNA was deposited in GenBank with accession no. AY167041.


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Fig. 1.   Cloning strategy. An initial ~900-bp fragment was amplified from cDNA from winter flounder RBCs by using primer pair 1 based on the sequences of the Na+/H+ exchanger beta -NHE and human and Amphiuma NHE1. Another ~1,000 bp upstream to this fragment was obtained by using primer pair 2. The remainder of the open reading frame (ORF) was obtained by using primer pair 3 (designed from the fragment obtained with primer pair 2 and the start of the trout beta -NHE1 ORF) and further amplified with primer pair 4. The 5' and 3' untranslated regions (UTRs) were obtained by rapid amplification of cDNA ends (RACE), with gene-specific primers (GSP) corresponding to regions in the winter flounder NHE1 ORF. The sequences of the primers used are given below the figure. paNHE1, Pseudopleuronectes americanus NHE1; fw, forward; rv, reverse; UPM, universal primer mix.

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 isoforms beta , 2, 3, and 4 (13). Rabbit polyclonal antibody XB-17 (kindly provided by Dr. M. Musch, University of Chicago) was raised against a fusion protein containing the cytoplasmic region of human NHE1 (amino acids 631-746). Polyclonal antibody 666 (a kind gift from Dr. J. Claiborne, Georgia Southern University) was produced against a fusion protein containing amino acids 528-648 of rat NHE3.

Immunoblotting. 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+, Cl-, and H2O in winter flounder RBCs was carried out essentially as previously described (10). All experiments were conducted at room temperature (20-25°C) and atmospheric PO2. After preparation and incubation as described above, cell suspensions at 10% hematocrit were pelleted by mild centrifugation (40 s, 1,800 g), the supernatant was removed by aspiration, and at time 0 the cells were resuspended in the desired flux medium. At the time points indicated, samples were taken by removal of 400-µl aliquots to washed, preweighed, narrow microcentrifuge tubes (E & K Scientific, Campbell, CA) for centrifugation (4 min, 16,750 g) and careful separation of the resulting cell pellets and supernatants. Cell pellet wet weights were noted, and the cells were subsequently lysed by mechanical disruption in 250 µl of lysis buffer (40 mM ZnSO4 and 4.6 mM MgSO4). The lysates were centrifuged 2 × 15 min at 16,750 g, the supernatants were stored for analysis, and the pellets were dried to constant weight (minimum of 18 h at 60-70°C). In each experiment, corrections for extracellular space were performed by parallel analysis of sets of samples (double determination) suspended in, respectively, Na+-free and Na+-containing isotonic medium. Data were then corrected by using the assumption that the difference in Na+ content between the Na+-free and Na+-containing samples reflects the contribution from trapped medium. This procedure has been found to correlate well with the [14C]polyethylene glycol method for extracellular space correction used in earlier studies (10). Cell H2O content was determined as the difference between pellet wet and dry weight, corrected for trapped medium. Cell Na+ and K+ content were determined by flame photometry (Instrumentation Laboratories model 443, Boston, MA). Cell Cl- content was determined by coulometric titration with silver ions by using a Buchler chloridometer (Searle Diagnostics, Fort Lee, NJ). Supernatant Na+, K+, and Cl- content were also determined, and these data were used in the corrections for trapped medium. Data are presented as means ± SE.

Measurements 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).


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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 beta -NHE (7, 36, 49). An additional 1 kb upstream was obtained by using primers based on the first fragment and on the European flounder cDNA sequence. The 5' and 3' UTR sequences were obtained by using the RACE procedure, with gene-specific primers based on regions in the predicted ORF of the winter flounder NHE. The 3' RACE reaction resulted in several distinct bands, the sequencing of which revealed the presence of three partially different 3' UTRs. The 5' UTR and ORF were identical except for three silent dimorphisms within the ORF at positions 1,224 (C or T), 1,350 (A or G), and 1,974 (A or G). Thus the deduced amino acid sequence was identical in all cases, and only the longest transcript will be dealt with in the present study (however, see DISCUSSION). Figure 2 shows the full-length, 3,690-bp cDNA and the deduced amino acid sequence. The ORF spans 2,340 bp and is thus predicted to code for a protein of 779 amino acids, corresponding to a calculated molecular weight of 86.6 kDa, not taking into account glycosylation. The translation initiation sequence, AAAATGG, is positioned 79 bp from the 5' end of the obtained cDNA and corresponds well to the Kozak sequence A/GCCATGG, where the A or G in position -3, and the G in position +4 are most critical (29). In the 3' UTR, a poly-A signal (AATAAA; Ref. 56) is positioned 1,214 bp downstream from the translation termination codon (TGA), followed after another 20 bp by an ~27-bp-long poly-A stretch.


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Fig. 2.   Nucleotide sequence and deduced amino acid sequence for NHE1 from winter flounder red blood cells (RBCs). The position of the translation initiation codon and Kozak sequence (AAAATGG), the translation termination codon (TGA), and the polyadenylation signal (AATAAA) are indicated in red. See text for details.

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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Fig. 3.   Relation of paNHE1 to other NHE proteins. A: cladogram illustrating the sequence distance between the paNHE1 protein and other NHEs. The cladogram was generated by calculation of genetic distances on the basis of the estimated number of substitutions per 100 amino acids by using the Kimura protein distance correction method (GCG Evolutionary analysis software). B: alignment of the paNHE1 amino acid sequence with those of trout beta -NHE and human and Amphiuma NHE1. The alignment was created by using GCG software. Stars indicate 100% identity across the four species. The 12 probable transmembrane (TM) regions and the region likely to "dip" into the membrane from the extracellular side are underlined. Potential N-glycosylation sites and protein kinase consensus sequences were detected by using PROSITE and are shown in, respectively, black bold (N-glycosylation), red [protein kinase A/protein kinase G (PKA/PKG)], and blue [protein kinase C (PKC)] (overlap between the S503 PKA/PKG and PKC sites shown in magenta).

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 beta -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 beta -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 beta -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 beta -NHE (corresponding to S481 and S648, respectively, in the beta -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 beta -NHE, NHE2, NHE3, or NHE4 (see Ref. 13). A polyclonal NHE1 antibody (XB-17) recognized a band of similar size, whereas no bands were detected in the membrane fractions from winter flounder RBCs using an antibody against NHE3 (not shown).


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Fig. 4.   Presence and subcellular localization of paNHE1 in winter flounder RBCs. A: membrane fractions of winter flounder RBCs were prepared as described in MATERIALS AND METHODS, separated on 10% SDS-polyacrylamide gels, and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking of unspecific binding, membranes were labeled with a mouse monoclonal NHE1 antibody, 4E9 (1:10,000), followed by the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody and visualization by enhanced chemiluminescence. The blot shown is representative of 3 independent experiments. In the absence of primary antibody, labeling was essentially undetectable (n = 3). The polyclonal NHE1 antibody XB-17 labeled a band of similar size, whereas no labeling could be detected with Ab 666, an antibody against NHE3 (not shown). B: RBCs were fixed in 3% paraformaldehyde as described in MATERIALS AND METHODS, washed in PBS with 0.5% Triton X-100, permeabilized in this medium for 15 min, blocked (3% BSA in PBS-0.5% Triton X-100), incubated with primary antibody (XB-17 at 1:100 in blocking buffer) for 1 h at room temperature, washed, and incubated with FITC-conjugated goat anti-rabbit IgG at 1:200 in the blocking buffer for 1 h, followed by extensive washing. Cells were transferred to no. 1 cover slips and visualized with the 63×/1.4 NA oil DIC objective of an LSM 510 Zeiss confocal system. The image shown is a projected image produced with a stack of Z sections (7.2 µm) of a representative image area and is representative of n = 2 independent experiments. Only very faint and diffuse labeling could be seen in control cells labeled with secondary antibody only (right; n = 2 independent experiments).

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<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. The parallel operation of these exchangers in conjunction with the activity of carbonic anhydrase leads to net uptake of NaCl and osmotically obliged water. In another common mechanism of RVI, ion and water uptake is the result of shrinkage-induced activation of Na+,K+,2Cl- 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<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger, the increase in Cl- 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<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. It should be noted that paNHE1 appears to be unique among NHE1 isoforms in that it is resistant to inhibition by the commonly used NHE inhibitors amiloride, 5'-(N-ethyl-N-isopropyl)amiloride (EIPA), and HOE 694 at doses up to 100 µM (EIPA, HOE 694) or 1 mM (amiloride) (S. A. King, R. R. Rigor, S. F. Pedersen, Z. Zhuang, and P. M. Cala, unpublished observations; Ref. 44). This property of paNHE1, which could be a valuable tool in the delineation of regions involved in inhibitor binding and ion translocation, is presently under investigation.


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Fig. 5.   Identification of the membrane transport proteins involved in RVI in winter flounder RBCs. A and B: cells were preequilibrated in isotonic medium as described in MATERIALS AND METHODS, followed by a further 2 × 20-min incubation with or without DIDS (10 µM) to inhibit the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. All cells were subsequently washed in isotonic medium containing 1 mM ouabain. At time 0, cells were exposed to isotonic (360 mosM) or hypertonic (540 mosM) medium, in the continued presence of ouabain, and aliquots were removed at the times indicated for determination of the cellular content of Na+, K+, and Cl-. Data are means ± SE of 3 independent experiments. C: RBCs were preequilibrated, treated with or without DIDS, and washed in 1 mM ouabain, as in A. Cells were subsequently transferred to poorly buffered isotonic or hypertonic medium, and extracellular pH (pHo) was monitored over time. The experiment shown is representative of 2 independent experiments.

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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Fig. 6.   Effect of hypertonic challenges of different magnitude on ion and water content in winter flounder RBCs. RBCs were preequilibrated for several hours in isotonic medium, and, immediately before initiation of the flux experiments, washed twice in isotonic medium containing 1 mM ouabain. All flux media contained 1 mM ouabain. At time 0, cells were diluted at 10% hematocrit in isotonic medium (open circle ), or media of 1.1 (), 1.5 (), or 2.0 (black-triangle) times isotonic osmolarity. Aliquots were removed at time points in the range of 0.7 to 90 min for determination of the cellular content of H2O, Na+, K+, and Cl-. The graphs illustrate the cellular content of H2O (A), Na+ (B), K+ (C), and Cl- (D) over time after hypertonic challenge. Data are means ± SE of 3 paired experiments. Parallel experiments were performed in media of 1.25 and 1.75 times isotonic osmolarity with similar results (n = 3; not shown).

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 beta -adrenergic agonist isoproterenol are shown in Fig. 7. Exposure to 1 µM isoproterenol elicited rapid increases in the cellular content of Na+, Cl-, and H2O. Similar findings were obtained by using forskolin (100 µM) to directly stimulate the adenylate cyclase (data not shown, n = 2). Corresponding dose-response curves for isoproterenol concentrations in the range from 1 nM to 100 µM are shown in Fig. 8. Significant increases in cellular NaCl and H2O content are only seen at isoproterenol concentrations of 100 nM or above, and maximal activation is reached at a concentration of 1 µM. In contrast to osmotic shrinkage, isoproterenol does elicit a distinct, albeit modest, decrease in cellular K+ content, evident at late (90 min) time points after stimulation (see DISCUSSION).


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Fig. 7.   Effect of isoproterenol on ion and water content in winter flounder RBCs. RBCs were preequilibrated and treated with 1 mM ouabain as described in the Fig. 6 legend. At time 0, the cells were diluted at 10% hematocrit in isotonic medium with (IP; ) or without (Ctrl; open circle ) 1 µM of the beta -adrenergic agonist isoproterenol, and aliquots were removed at the times indicated for determination of the cellular content of H2O (A), Na+ (B), K+ (C), and Cl- (D). Data are means ± SE of 3 experiments.



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Fig. 8.   Dose-response relationship for the isoproterenol-induced changes in cellular ion and water content in winter flounder RBCs. Experiments were carried out as described in the Fig. 7 legend, but with isoproterenol concentrations ranging from 1 nM to 100 µM. Data are shown as the cellular content of H2O (A), Na+ (B), Cl- (C), and K+ (D) as a function of the isoproterenol concentration (logarithmic axis) at 10 min () and 90 min (open circle ) after stimulation. Data are means ± SE of triplicate experiments.

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-, and H2O content, with only modest changes in K+ content. Figure 10 shows the corresponding dose-response curves for CL-A. Significant increases in NaCl and H2O content require CL-A concentrations of at least 50 nM, whereas 100 nM CL-A elicits maximal increases. Cellular ion content at time 90 min is similar after saturating doses of either isoproterenol or CL-A, with the most notable difference between the two stimuli being the somewhat slower time course of the CL-A mediated increase, as seen by comparing the fractional increases at times 10 and 90 min.


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Fig. 9.   Effect of the serine/threonine phosphatase inhibitor calyculin A (CL-A) on cellular ion and H2O content in winter flounder RBCs. RBCs were preequilibrated and treated with 1 mM ouabain as described in the Fig. 6 legend. At time 0, the cells were diluted at 10% hematocrit in isotonic medium with (CL-A; ) or without (Ctrl; open circle ) 100 nM CL-A, and aliquots were removed at the times indicated for determination of the cellular content of H2O (A), Na+ (B), Cl- (C), and K+ (D). Data are means ± SE of triplicate experiments.



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Fig. 10.   Dose-response relationship for the CL-A-induced changes in cellular ion and H2O content in winter flounder RBCs. Experiments were carried out as described in the Fig. 9 legend, but with CL-A concentrations of 10, 50, 100, and 500 nM. Data are shown as the cellular content of H2O (A), Na+ (B), Cl- (C), and K+ (D) as a function of the CL-A concentration, at 10 min () and 90 min (open circle ) after stimulation. Data are means ± SE of triplicate experiments.

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- content, whereas cellular K+ content was essentially unaffected, as seen for the individual stimuli (not shown).


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Fig. 11.   Additivity of the changes in cellular ion and H2O content in winter flounder RBCs after stimulation by osmotic shrinkage, cAMP, and CL-A. RBCs were preequilibrated and treated with 1 mM ouabain as described in the Fig. 6 legend. At time 0, the cells were diluted at 10% hematocrit in the indicated experimental medium, and aliquots were removed at the times indicated for determination of the cellular content of H2O, Na+, K+, and Cl-. Shown on the left are the Na+ (A) and H2O (C) content of isotonic control cells (open circle ) and cells stimulated in isotonic medium with either 10 µM isoproterenol (IP; ), 100 nM CL-A (), or 10 µM IP plus 100 nM CL-A (black-triangle). Shown on the right are the Na+ (B) and H2O (D) content of isotonic control cells (open circle ) and cells shrunk in medium of 1.5× isotonic osmolarity without () or with either 10 µM isoproterenol () or 100 nM CL-A (black-triangle). Data are means ± SE of 15 (isoproterenol), 12 (osmotic shrinkage), 6 (osmotic shrinkage plus isopoterenol), or 3 (CL-A, osmotic shrinkage plus CL-A, isoproterenol plus CL-A) experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 trout beta -NHE (7). NHE1 is predicted to have a secondary structure consisting of an NH2-terminal region of 10-12 TM domains necessary for ion translocation and a COOH-terminal cytoplasmic tail that is dispensable for ion translocation but important in the regulation of NHE1 function (9, 54). Hydropathy analyses of paNHE1 suggest that this exchanger has a structure similar to that recently proposed for human NHE1 (53), consisting of a short cytoplasmic NH2 terminus, an amphipathic region with 12 TM domains and a hydrophobic region between TM 9 and 10 dipping into the membrane from the extracellular side, and a COOH-terminal hydrophilic tail of ~300 amino acids. In Western blots of RBC membrane fractions, a NHE1-specific antibody recognized a prominent band of ~100 kDa, compared with the calculated mass of 86.6 kDa. In vivo glycosylation is probably responsible for the difference between calculated and apparent molecular mass, consistent with findings for other NHE1s (9, 54). Confocal imaging of fixed flounder RBCs showed that paNHE1 is primarily localized to the marginal band of the RBCs, consistent with a role in membrane transport.

Nature 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 Cl- content was abolished by inhibition of the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger with DIDS, whereas the increase in Na+ content was unaffected, and that shrinkage of DIDS-treated cells, but not of control cells, led to significant extracellular acidification. Had a Na+-K+-2 Cl- or Na+-Cl- cotransporter been operating, extracellular acidification would have occurred in the absence of DIDS (due to Cl- recycling through the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger), and this acidification would have been prevented, rather than exacerbated, by DIDS treatment. Taken together, these findings strongly indicate that RVI in flounder RBCs is mediated by the parallel activity of a NHE and a Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger. This is similar to the mechanism employed in Amphiuma RBCs (11) but inconsistent with RVI by other known mechanisms, such as Na+-K+-2 Cl- or Na+-Cl- cotransport, Na+ channels, or organic osmolyte uptake (24). The finding that the flounder RBC NHE appears to be insensitive to amiloride and its derivatives is a unique and interesting property of this transporter, which is presently under investigation.

Because 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 (Delta µNa+ - Delta 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<UP><SUB>−</SUB><SUP>3</SUP></UP> exchanger cannot serve as a dynamic H+ buffer.

Stimulation of the RBCs with the beta -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 a Delta 635 but not in a Delta 698 truncation mutant (4). It is noteworthy that although paNHE1 exhibits overall higher identity to the largely shrinkage-insensitive beta -NHE than to the NHE1s from human or Amphiuma, the region in paNHE1 that corresponds to amino acids 636-656 in human NHE1 exhibits 76% identity to this region in human NHE1 and only 62% to the corresponding region in beta -NHE. This is consistent with the notion that this region plays an important role in shrinkage-induced NHE1 activation and warrants future mutagenesis studies of the motif(s) in paNHE1 required for shrinkage-mediated activation.

Under 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 beta -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 beta -NHE. S503 is also shared by beta -NHE, but the corresponding site, beta NHE S481, does not appear to contribute to the cAMP-mediated activation of beta -NHE, because this activation is ablated in a Delta 559 mutant (6). In support of the notion that PKA-mediated phosphorylation of this motif plays a major role, the S648 motif of the beta -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 beta -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 beta -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, whereas beta -NHE1 is essentially shrinkage insensitive, although the rainbow trout is also capable of migration between freshwater and seawater.

Activation 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 beta -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 beta -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 beta -NHE and 65% to the human NHE1. Functionally, paNHE1 shares characteristics of both beta -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 beta -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<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger, their parallel operation results in net uptake of NaCl and osmotically obliged water. Activation of paNHE1 by cAMP is at least additive to that elicited by osmotic shrinkage, suggesting that these stimuli regulate paNHE1 by distinct mechanisms. Finally, exposure to the serine/threonine phosphatase inhibitor CL-A potently activates paNHE1, and this activation is also additive to that induced by shrinkage or cAMP.


    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.


    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<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger is greater in the more hypertonic condition, presumably due to a pHi-dependent increase in activity of this exchanger at alkaline pHi.

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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