HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 285/3/R526    most recent 00146.2003v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (16) Citing Articles via Google Scholar Google Scholar Articles by Nickerson, J. G. Articles by Moon, T. W. Search for Related Content PubMed PubMed Citation Articles by Nickerson, J. G. Articles by Moon, T. W.

MODEL ORGANISMS AND COMPARATIVE FUNCTIONAL GENOMICS

Activity of the unique -adrenergic Na⁺/H⁺ exchanger in trout erythrocytes is controlled by a novel ₃-AR subtype

Department of Biology, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5

Submitted 21 March 2003 ; accepted in final form 23 May 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
-Adrenoceptors (-ARs) are seven-transmembrane domain, G protein-coupled receptors that transduce the cellular effects of epinephrine and norepinephrine and play a pivotal role in the vertebrate stress response. This study reports the cloning and characterization of two previously unreported -ARs from the rainbow trout (Oncorhynchus mykiss). Phylogenetic analysis of amino acid sequences indicates that both -ARs are homologs of the mammalian ₃-AR. Analysis of tissue expression patterns indicates that one of these trout ₃-adrenoceptors (_3a-AR) is highly expressed in gill and heart, whereas the second (_3b-AR) is highly expressed by red blood cells (RBC). Expression of the _3b-AR in the RBC coupled with the finding of a single category of -AR binding sites on RBC membranes provides strong evidence for the control of the trout RBC -AR Na⁺/H⁺ exchanger (-NHE) activity by signaling through this _3b-subtype and not through a ₁-subtype as previously proposed. The RBC-specific trout _3b-AR exhibits binding characteristics that distinguish this receptor from each of the three pharmacologically defined categories of mammalian -ARs (₁-, ₂-, and ₃-AR). This study is the first to report the presence of a ₃-AR subtype in a fish species, and the proposal that the _3b-AR controls RBC -NHE activity represents a novel role for the ₃-AR subtype in vertebrates.

molecular sequence; tissue expression; evolutionary analysis; binding kinetics; -adrenoceptors

Despite their importance in the vertebrate stress response, our knowledge of -ARs from nonmammalian vertebrates is based on relatively few studies. This is particularly true at the molecular level, where only a handful of studies describe nonmammalian -ARs (5, 6, 24, 46). In certain organisms, novel tissue -AR responses have evolved that assist these organisms in coping with a specific set of environmental/physiological stressors. Owing to their unique characteristics, some of these novel tissue -AR responses from nonmammalian species have attracted considerable attention. For example, the -AR Na⁺/H⁺ exchanger (-NHE) system found in the rainbow trout red blood cell (RBC) (3) has been extensively studied physiologically, and this system has become one of the most well-characterized nonmammalian -AR-mediated responses (for reviews, see Refs. 22, 25). In trout, catecholamines are able to augment hemoglobin/oxygen-binding affinity by increasing RBC intracellular pH through typical -AR signaling (3, 13, 25, 32). Activation of the trout RBC -AR leads to accumulation of cAMP and activation of PKA that in turn activates -NHE by phosphorylation (2, 3, 19). Once activated, the -NHE extrudes H⁺ from the RBC in exchange for Na⁺, resulting in alkalization of the cytoplasm. This increase in RBC intracellular pH, in turn, enhances the affinity of hemoglobin for O₂ and allows for increased oxygen transport by the blood (for reviews, see Refs. 25, 27, 31). The results of previous pharmacological studies of this novel system suggested that a ₁-AR subtype controls the activity of the -NHE (41). Much of the attention directed at this trout RBC system has focused on characterizing its novel -NHE, which is the only known form of NHE to be activated by a cAMP/PKA pathway (3). Despite the critical role played by the -AR, no molecular data exist describing the RBC -AR, and the previous ₁-subtype classification of the trout RBC receptor remains uncorroborated at the molecular level.

Thus the aim of this study was to broaden our knowledge of the rainbow trout -AR gene family in general and to provide molecular evidence for the specific -AR subtype in the trout RBC. Here we present the characterization of two previously unreported -AR genes from the rainbow trout. Phylogenetic analysis indicates that these two newly cloned trout -ARs are homologous to the mammalian ₃-AR, and thus we call these receptors rainbow trout _3a- and _3b-ARs. Analysis of rainbow trout _3a- and _3b-AR tissue expression patterns reveals that the RBC specifically expresses _3b-AR, suggesting that -NHE activity in the trout RBC is modulated by signaling through a ₃-AR subtype and not a ₁-AR subtype as previously hypothesized. This is the first report of ₃-ARs in fish, and the proposed control of the trout RBC -NHE system by _3b-AR signaling represents a novel role for ₃-ARs in vertebrates.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Experimental animals. Rainbow trout, Oncorhynchus mykiss, weighing between 200 and 400 g, were obtained from Linwood Acres Trout Farm (Campbellcroft, Ontario, Canada) and were held indoors in large fiberglass tanks supplied with dechlorinated city of Ottawa tap water that was maintained at 13°C. Fish were allowed to acclimate to the aquarium for at least 3 wk before experimentation. Fish were maintained on a 12:12-h light/dark photoperiod and fed daily to satiation with a commercial salmonid diet until 24 h before experimentation.

Isolation of RNA. For RT-PCR and RNase protection assays (RPAs), trout were perfused through the heart with 1 liter of saline (0.9% NaCl) to flush blood from the tissues before tissue isolation. Total cellular RNA was isolated from fresh tissues of the rainbow trout using Trizol reagent (GIBCO BRL). RNA concentrations and quality were verified using spectrophotometry and agarose gel electrophoresis. Aliquots of RNA to be used in RT-PCR, RPAs, or quantitative (Q)-PCR were treated with DNase1 DNA free kit (Ambion Austin, TX) before use.

Amplification of rainbow trout _3a- and _3b-AR cDNA. An initial set of trout _3a- and _3b-AR clones spanning the first to sixth transmembrane domains (750 bps) were amplified using a nested RT-PCR strategy. Oligo-p(dT)₁₅ primed cDNA was synthesized using the First Strand cDNA Synthesis Kit for RT-PCR (Roche Molecular Biologicals). A preliminary round of PCR amplification was performed using the degenerate primers AdrUni 5' and AdrUni 3' (Table 1), followed by a second round of amplification using the nested degenerate primers BetaUni 5' and BetaUni 3' (Table 1). All putative -AR clones were sequenced using the Big Dye Terminator Cycle Sequencing kit version 3.0 (PE Applied Biosystems) and standard M13 forward (-20) and reverse sequencing primers. The sequence of these clones was then used to design gene-specific primers for 5'- and 3'-RACE of both trout _3a- and _{3 b}-ARs.

View this table: [in this window] [in a new window]   Table 1. Primers used for amplification of various fragments of the rainbow trout 3a - and 3b-ARs

RACE PCR. The 5'- and 3'-RACE System for Rapid Amplification of cDNA Ends, version 2 (GIBCO BRL), was used to amplify the 5'- and 3'-ends of the trout _3a- and _3b-AR cDNAs. In the 5'-RACE protocol, trout _3a- and _3b-AR gene-specific primers _3a-GSP1 and _3b-GSP1 (Table 1) were used to prime cDNA synthesis of trout _3a- or _3b-ARs separately. These cDNAs were then used as template in an initial round of PCR amplification using a second set of primers specific for either trout _3a-AR (_3a-GSP2) or _3b-AR (_3b-GSP2) (Table 1) and the 5'-amplification primer provided with the kit. A 5-µl aliquot of the initial PCR amplification was then used as a template for a second round of PCR using nested primers specific for either trout _3a-AR (_3a-GSP3) or _3b-AR (_3b-GSP3) (Table 1) and the abridged universal amplification primer (AUAP) provided with the kit.

Synthesis of cDNA for 3'-RACE was primed with the 3'-amplification primer provided in the kit. A first round of PCR amplification was performed using trout _3a- or _3b-AR gene-specific primers, _3a-GSP4 or _3b-GSP4, respectively (Table 1), and AUAP. A second round of semi-nested PCR amplification using trout _3a- or _3b-AR gene-specific primers _3a-GSP5 or _3b-GSP5 (Table 1) and AUAP was then carried out.

The complete coding region of the trout _3b-AR was PCR cloned using nondegenerate primers, rainbow trout _3b complete coding sequence (CDS), designed from the sequence of the _3b-AR 5'- and 3'-RACE clones.

All PCR amplifications described above used the following regimen of denaturing, annealing, and extension: 1 x 2 min at 94°C; 30 x 30 s at 94°C, 30 s at 45-60°C, 1 min at 72°C; and 1 x 10 min at 72°C. Annealing temperatures varied from 45 to 60°C depending on the primer sets being used (Table 1).

Sequence analyses. The rainbow trout _3a- and _3b-AR amino acid sequences (derived by conceptual translation of nucleotide sequences) were aligned with GenBank sequences of various -AR subtypes from selected organisms (Table 2) using default settings in CLUSTAL W version 1.8 (42). Maximum likelihood phylogenetic analysis was performed using PUZZLE version 4.0.2 (39). The following program settings were used: quartet puzzling tree search, compute exact quartet likelihood, 1,000 puzzling steps, use the amphioxus dopamine/-AR sequence as outgroup, branch lengths are not clocklike, JTT model of substitution, amino acid frequencies were estimated from the data set, and the model of rate heterogeneity was 1 invariable + 8 gamma rates. The trout _3a- and _3b-AR sequences were analyzed for the presence of gene conversion events using GENECONV version 1.70 (35). Because GENECONV analyses work best with more than two sequences, this analysis was performed on a data set composed of the trout _3a-, trout _3b-, and puffer fish ₃-AR sequences (8). The analysis was performed using a g scale of two to allow for some mismatches in the converted regions (35).

RPA. The expression patterns of the rainbow trout _3a-and _3b-AR genes were determined using an RPA, RPAIII (Ambion). Eight tissues were assessed: gill, heart, kidney, liver, red muscle, white muscle, blood, and spleen. The template used to synthesize the probes used in RPA experiments was obtained by PCR amplification of plasmid clones of the trout _3a- or _3b-ARs. These probes included the third intracellular loop region and corresponded to nucleotides 804 to 958 for rainbow trout _3a-AR and 747 to 952 for rainbow trout _3b-AR (Fig. 2). The primers used to amplify probe template incorporated the promoter sequences for T7 and SP6 RNA polymerases (Table 1) so that antisense or sense RNA probes could be transcribed. Radiolabeled antisense RNA probes were transcribed using MAXIscript (Ambion) with T7 RNA polymerase and ³²P-UTP (Amersham Pharmacia). Full-length probes were isolated from denaturing 4% polyacrylamide, 8 M urea gels. Approximately 4.2 x 10⁴ cpm of probe was hybridized to 20 µg of total RNA for 16 h at 42°C. Nonhybridized transcripts were digested with 0.4 units of RNase A and 15 units of RNase T1 at 37°C for 90 min. Protected fragments were resolved on denaturing 6% polyacrylamide, 8 M urea gels that were dried and subjected to autoradiography.

View larger version (73K): [in this window] [in a new window]   Fig. 2. Alignment of rainbow trout 3a- and 3b-AR nucleotide sequences from the NH2 to the COOH terminal. NH2-terminal extracellular tail extends from position 1 to 174 of both 3a- and 3b-AR, whereas the COOH-terminal tail extends from position 1113 to 1284 of 3a and position 1107 to 1434 of 3b. Gray highlight, position of the deduced transmembrane domains; +, synonymous nucleotide substitutions; *, nonsynonymous substitution and indel (appearing above the substituted position); indel, insertion/deletion. Region of gene conversion extends from position 1 to 245. Arrows indicate the positions of the 3'-end of the primers used to amplify the RNase protection assay (RPA) probe template.

Quantification of -AR mRNA levels. cDNA was synthesized from 1 to 2 µg total RNA using random hexamer primers and Superscript reverse transcriptase (GIBCO BRL). -AR mRNA levels were assessed by QPCR on duplicate samples of cDNA (1 µl) using a Hot StarTaq Master Mix kit (Qiagen) and a Stratagene MX-4,000 multiplex QPCR system. CYBR Green (Molecular Probes) and ROX (Stratagene) were used as DNA and reference dyes, respectively. The PCR conditions (final reaction volume = 20 µl) were as follows: cDNA template = 1.0 µl; forward and reverse primer = 150 pmol/l; Mg²⁺ concentration = 2.0 mmol/l; CYBR green = 1:50,000 final dilution; ROX = 1:30,000 final dilution; dNTPs = 200 µmol/l. The annealing and extension temperatures were 58 and 72°C, respectively. Gene-specific primers for rainbow trout ₂-AR (QPCR ₂-AR), rainbow trout _3b-AR (QPCR _3b-AR), and trout -actin (QPCRact) were designed using Primer3 software (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi) (Table 1). The specificity of the primers was verified by cloning and sequencing of the amplified products. To ensure that CYBR green was not being incorporated into primer dimers or nonspecific amplicons during the QPCR runs, PCR products were analyzed by agarose gel electrophoresis in initial experiments; single bands of the expected size were obtained in all instances. Furthermore, the construction of CYBR green dissociation curves after completion of 40 PCR cycles revealed the presence of single amplicons for each primer pair. Relative expression of mRNA levels was determined (using actin as an endogenous standard) by a modification of the delta-delta Ct method (30). Amplification efficiencies were determined from standard curves generated by serial dilution of plasmid DNA.

Kinetic analysis of receptor binding. Kinetic analysis of the rainbow trout _3b-AR was carried out in intact trout RBCs using the hydrophilic -AR ligand [(-)-4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-³H] benzimidazol-2-one][³H]CGP-12177 ([³H]CGP; Amersham, specific activity 46.0 Ci/mmol). Previous studies have characterized CGP-12177 as an antagonist of mammalian ₁- and ₂-ARs and as an agonist of mammalian ₃-ARs (34). Whole blood was diluted 10-fold in Hanks' buffered saline (in mM: 136.7 NaCl, 5.4 KCl, 0.8 MgSO₄, 0.33 NaH₂PO₄, 0.44 KH₂PO₄, 5.0 HEPES, 5.0 HEPES-Na, 1.0 NaHCO₃, and 0.06 L-ascorbic acid, pH 7.6). Fifty microliters of cells were incubated for 45 min at room temperature in a final volume of 150 µl in the presence of a saturating concentration of [³H]CGP (5 nM) alone or with increasing concentrations of selective and nonselective agonists and antagonists. With the exception of CL-316,243 (a gift from Dr. Jean Himms-Hagen, Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa), all agonists and antagonists used were purchased from Sigma (St. Louis, MO). Incubations were carried out in dim light and aluminum foil was used to cover the ligand-containing microcentrifuge tubes to prevent photodegradation. Assays were terminated using four washes of ice-cold 0.9% NaCl and a cell membrane harvester (Brandel 24R). Glass fiber filters (#32, Schleicher and Schuell) were placed in scintillation vials containing 4 ml scintillation cocktail (Safety-Solve; RPI, Mount Prospect, IL). Radioactivity was determined using a Packard 2500TR liquid scintillation counter after 24 h of incubation in the dark.

Physiological assessment of RBC -NHE activity. Trout were anesthetized in a solution of ethyl-P-amino-benzoate (benzocaine; Sigma; final concentration 2.4 x 10^-4 mol/l) and placed onto an operating table, where the gills were continuously irrigated with aerated anesthetic solution. An in-dwelling polyethylene cannula (Clay-Adams PE-50 polyethylene tubing; internal diameter 0.580 mm, outer diameter 0.965 mm) was implanted into the dorsal aorta (36) to permit blood sampling. Fish were revived on the operating table by irrigation of the gills with aerated water, then transferred to individual opaque acrylic experimental chambers (volume = 3 liters) supplied with aerated, flowing water. Cannulas were flushed daily with freshwater teleost saline (44) containing 50 U/ml ammonium heparin (Sigma).

Blood was withdrawn from the dorsal aortic cannula of fish and pooled to obtain a sufficient volume of blood for a single experiment. In practice, 6.0-8.0 ml was obtained from two fish and this was usually sufficient to yield enough blood for an entire experimental series. Blood sampling was halted immediately at the first sign of agitation or struggling. Because of the possibility that the blood contained higher than normal levels of circulating catecholamines, the pooled sample was gassed with O₂ for 2 min to increase the rate of catecholamine degradation. The blood was then stored on ice in 25-ml round-bottom tonometer flasks (final heparin concentration = 50 U/ml) for 4-6 h before experimentation. A previous report showed that catecholamines decay with a half-life of 24 min when stored at 15°C under similar conditions (41), and thus it is likely that the catecholamine levels were low in the blood at the time of experimentation.

The extent of activation of RBC -AR-mediated Na⁺/H⁺ exchange was assessed by real-time monitoring of whole blood pH before and after addition of AR agonists and/or antagonists (29). Blood (1.0 ml) was transferred to a glass tonometry flask (Eschweiler; 5 ml vol) immersed in a 13°C water bath. Blood was then equilibrated by shaking for 20 min with a hypoxic (PCO₂ = 0.25 kPa; PO₂ = 1.5 kPa, remainder N₂) gas mixture. After 20 min, the blood was pumped (peristaltic pump; 0.4 ml/min) through a temperature-controlled chamber (13°C) housing a combination pH electrode (Metrohm) and then returned to the tonometry flask. To prevent blood from clotting in the tubing and electrode chambers, the loop was rinsed before each experiment for 10-15 min with heparinized (540 U/ml) saline. On achieving a stable baseline pH (usually within 5 min), the shaking was stopped momentarily (<10 s) to allow injection (20 µl) of adrenergic agonists or saline (controls) into the equilibrated blood; whole blood pH was then monitored for an additional 15 min. The maximal fall in pH (usually achieved within 5 min of agonist addition) was used as an index of Na⁺/H⁺ exchange activity (29). Analog pH data output from a Radiometer PHM 73 blood gas analyzer was converted to digital data and stored by interfacing with a data-acquisition system (Biopac Systems) using Acknowledge data-acquisition software (sampling rate set at 30 Hz) and a Pentium personal computer. In experiments to show antagonist inhibition of the isoproterenol-stimulated decreased in blood pH, various adrenergic antagonists (20 µl) were added to the blood at the beginning of the 20-min equilibration period before addition of isoproterenol. Detection of statistically significant differences between the changes in blood pH elicited by isoproterenol alone vs. isoproterenol + antagonists was tested using a one-way ANOVA (Sigma Stat version 2.0, SPSS). The agonists isoproterenol (nonselective -AR), clenbuterol (selective mammalian ₂-AR), dobutamine (selective mammalian ₁-AR), and BRL (selective mammalian ₃-AR) were used at final concentrations of 10^-7 mol/l. The antagonists atenolol (selective mammalian ₁-AR), ICI (selective mammalian ₂-AR), and CGP (a mixed mammalian ₁-/₂-AR) were used at a final concentration of 10^-5 mol/l.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Maximum likelihood analysis of aligned vertebrate -AR amino acid sequences including the rainbow trout _3a- and _3b-ARs produced a tree with three major groups corresponding to the three pharmacologically defined -AR subtypes and placed the trout and puffer fish ₃-ARs at the base of the mammalian/avian ₃-AR group with strong statistical support (97%) (Fig. 1). Phylogenetic analysis also indicated that vertebrate ₁- and ₃-AR subtypes are more closely related to one another than either is to the ₂-AR subtype, although the support value for this relationship is relatively low (67%).

View larger version (17K): [in this window] [in a new window]   Fig. 1. Phylogenetic relationships of vertebrate -adrenoceptor (AR) subtypes inferred from maximum likelihood analysis of -AR amino acid sequence alignment. Three major groups correspond to the well-established mammalian -AR subtype nomenclature. Horizontal branch lengths are scaled to represent the relative number of amino acid substitutions occurring along a branch, and numbers at the nodes represent percent support values out of 1,000. Nodes without numbers represent relationships where the support values were <50%. Amphioxus dopamine/-AR sequence was used as the outgroup.

The rainbow trout _3a- and _3b-AR genes encode proteins of 429 and 477 amino acids, respectively. Comparison of the rainbow trout _3a- and _3b-AR sequences reveals a high degree of sequence conservation at both the amino acid and nucleotide levels and the presence of an additional 48 amino acids in the cytoplasmic tail of the rainbow trout _3b-AR relative to rainbow trout _3a-AR (Fig. 2). In addition, a gene conversion event at the 5'-end of the rainbow trout _3a- and _3b-AR genes was detected using GENECONV (35). This conversion event is situated between bases 1 and 245 and is strongly supported (P = 0.01). In this region of 245 bases, there are only four nucleotide substitutions between _3a-AR and _3b-AR (1.6% substitution), whereas in the remaining 1039 bases there are 108 nucleotide substitutions between the two genes (10.4% substitution).

In a multiple alignment of vertebrate -AR amino acid sequences, the trout ₃-ARs showed the highest degree of sequence identity with one another (84%) (results not shown). The rainbow trout _3a-AR amino acid sequence has an average identity of 54.6%, 51.2%, and 52.8% to other vertebrate ₁-, ₂-, and ₃-ARs, respectively, whereas rainbow trout _3b-AR shows an average amino acid identity of 54.2%, 52.2%, and 52.0% to other vertebrate ₁-, ₂-, and ₃-ARs, respectively. The highest levels of sequence conservation occurred within the seven transmembrane domains, whereas the amino terminal extracellular tail, third intracellular loop, and carboxy terminal cytoplasmic tail regions were the most variable (results not shown).

Tissue-specific expression patterns of the rainbow trout _3a- and _3b-ARs were determined using RPA. Rainbow trout _3a- and _3b-AR gene-specific probes for RPA were designed to span the third intracellular loop, a region of relatively high sequence variability between the rainbow trout _3a- and _3b-ARs (Fig. 2). Expression of rainbow trout _3a- and _3b-ARs was examined in eight tissues. RPA experiments showed high levels of _3a-AR mRNA in gill and heart and low levels in red muscle. There was no detectable _3a-AR expression in kidney, liver, white muscle, blood, or spleen after 8 h of autoradiography (Fig. 3). Rainbow trout _3b-AR mRNA was highly expressed only in blood (Fig. 3). The specific expression by the blood of high levels of _3b-AR mRNA, suggested by RPA analysis, was further verified by QPCR experiments measuring _3b-AR mRNA levels relative to -actin in the eight tissues studied (Fig. 4). To validate this QPCR approach, the tissue distribution of trout ₂-AR mRNA was also examined (Fig. 4). Results of the ₂-AR QPCR experiment were consistent with those of a previous study demonstrating a broad tissue distribution of ₂-AR mRNA in trout tissues (24). Identity of the amplified fragments was verified by sequencing in each set of QPCR experiments. Northern blot analysis of trout blood total RNA using a _3b-AR-specific probe demonstrated the presence of a 3.6-kb band in blood (results not shown). The _3b-AR-specific probe used in the Northern blot experiment corresponded to the amplicon in the QPCR experiments.

View larger version (66K): [in this window] [in a new window]   Fig. 3. RPA showing the expression pattern of the rainbow trout 3a-AR (A) and 3b-AR (B) gene in 8 different tissues. Gene-specific probes (see RESULTS) were hybridized to tissue total RNA. A fully protected fragment of 154 bases representing the 3a-AR is present in gill, heart, and red muscle. A fully protected fragment of 205 bases representing the 3b-AR is present in blood. A second smaller band caused by premature termination of probe transcription is also present in blood.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Quantitative PCR showing the expression pattern of rainbow trout (RbT) 3b-AR (A) and 2-AR (B) relative to -actin in 8 trout tissues. Abundance of 3b-AR mRNA detected in each tissue is expressed relative to that detected in blood (A). Highest levels of 3b-AR expression are seen in the blood. Abundance of 2-AR mRNA detected in each tissue is expressed relative to that detected in liver (B).

Pharmacological characteristics of the rainbow trout _3b-AR were determined by competitive binding assays performed on intact trout RBCs using the -AR ligand [³H]CGP. Preliminary experiments to determine association kinetics found a disassociation constant of 1.07 nM for [³H]CGP binding. Among the nonselective -AR agonists, isoproterenol inhibited [³H]CGP binding the most effectively with an inhibition constant (K_i) of 7.14 ± 0.78 µM(n = 5) followed by Epi and NE (Fig. 5). Neither Epi nor NE produced greater than 50% inhibition of [³H]CGP binding at the concentrations used; therefore, no K_i values could be estimated. Displacement of [³H]CGP binding by -AR subtype-selective agonists indicated that only the ₂-AR-specific agonist clenbuterol produced significant displacement of [³H]CGP binding with a K_i of 809 ± 168 nM (n = 5) (Fig. 5). None of the remaining -AR-selective agonists, dobutamine, procaterol, BRL-37344, or CL-316,243, displaced [³H]CGP. Among the ₁/₂-selective antagonists, the ₂-AR-selective ICI-118,552 inhibited [³H]CGP binding most effectively, with a K_i of 478 ± 118 nM (n = 4) followed by BAAM (₂ > ₁; 1.79 ± 0.32 µM, n = 6) and atenolol (₁-selective; K_i = 478 ± 118 µM, n = 4; Fig. 6). Both -AR-nonselective antagonists propanolol and nadalol inhibited [³H]CGP binding to trout RBCs with K_i values of 1.40 ± 0.33 (n = 6) and 55.3 ± 28.7 nM (n = 3), respectively (Fig. 6).

View larger version (14K): [in this window] [in a new window]   Fig. 5. Competitive displacement of (-)-4-(3-t-butylamino-2-hydroxypropoxy)-[5,7-3H] benzimidazol-2-one][3H]CGP-12177 ([3H]CGP) bound to rainbow trout red blood cell (RBC) 3b-ARs by the endogenous hormones epinephrine (Epi), norepinephrine (NE), and the synthetic agonist isoproterenol (Iso) (A) and the subtype-selective synthetic agonists dobutamine (Dob), procaterol (Proc), clenbuterol (Clen), BRL-37344, and CL-316,243 (B).

View larger version (15K): [in this window] [in a new window]   Fig. 6. Competitive displacement of [3H]CGP from rainbow trout RBC 3b-ARs by 1- and 2-AR subtype-selective synthetic antagonists atenolol (ATL), BAAM, and ICI-118,551 (A) and the -nonselective synthetic antagonists CGP-12177, propranolol (Prop), and nadalol (Nad) (B).

The pharmacological characteristics of the trout _3b-AR were further investigated by examining the ability of various mammalian -AR agonists and antagonists to activate/inhibit the rainbow trout RBC -AR/-NHE response in vitro. Activation of the RBC -NHE was measured as a decrease in the extracellular pH of the blood. Addition of the nonselective -AR agonist isoproterenol to the blood produced the largest change in pH, followed by the ₃-AR selective BRL (Fig. 7). Dobutamine and clenbuterol, which are ₁-AR- and ₂-AR-selective agonists, respectively, did not significantly decrease blood pH (Fig. 7). Antagonist inhibition of the isoproterenol-stimulated decrease in blood pH was highest using the ₂-AR-selective ICI (63.5% inhibition). Both the ₁-AR-selective antagonist atenolol and the mixed ₁-/₂-AR antagonist CGP failed to significantly inhibit the isoproterenol-stimulated decrease in blood pH (Fig. 7). Interestingly, the isoproterenol response of trout RBCs was inhibited most effectively by clenbuterol (77.9% inhibition) (Fig. 7), a selective agonist of mammalian ₂-ARs. The level of inhibition produced by clenbuterol was higher than that produced using atenolol and ICI together (70.6%; Fig. 7).

View larger version (16K): [in this window] [in a new window]   Fig. 7. Impact of -AR agonists and antagonists on trout whole blood proton extrusion as measured by changes in whole blood pH (pH). -AR antagonists were evaluated on the basis of their ability to inhibit the isoproterenol-induced decrease in blood pH. Aten, atenolol; , agonists that induced significant changes in whole blood pH (t-test); , antagonists that significantly inhibited the isoproterenol response (ANOVA on ranks).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
Maximum likelihood analysis of aligned vertebrate -AR amino acid sequences produced a tree with three major groups corresponding to each of the three pharmacologically defined -AR subtypes and placed the trout _3a-, _3b-, and puffer fish ₃-ARs in the mammalian/avian ₃-AR group (Fig. 1). The location of the trout and puffer fish ₃-ARs within this group (with strong statistical support) indicates that these fish receptors are homologous to mammalian ₃-AR (Fig. 1). This is the first study to demonstrate the presence of a ₃-AR in fish. Although the distribution of the ₃-AR in mammalian tissues has recently expanded beyond adipose tissue, the major role of the ₃-AR subtype in most mammals remains the mediation of the adipose tissue thermogenic response (1, 12, 17, 21, 23). The presence of ₃-ARs in trout and puffer fish, which are not expected to possess a thermogenic response, is of interest as it implies a novel role for this receptor subtype in fish.

Comparison of rainbow trout _3a- to _3b-AR sequences indicates that overall these genes are highly similar at both the amino acid and nucleotide levels (Fig. 2). The apparent lack of sequence divergence between the extracellular tails of the two trout ₃-AR sequences is explained by the presence of a gene conversion event extending from position 1 to 245 between these two genes (Fig. 2). Gene conversion occurs between regions of DNA with similar, but not identical, sequences and is common among members of a gene family (4). Previous studies of mammalian -ARs suggest that the extracellular tail may not play a significant role in receptor function (7, 37, 43). The presence of a gene conversion event between two -ARs in this region is therefore not likely to adversely affect receptor function. The possibility that the close phylogenetic relationship of the trout _3a- and _3b-AR genes may be due to this gene conversion event detected between the two sequences can be ruled out because exclusion of the converted region from the phylogenetic analysis did not alter tree topology (result not shown). This result suggests that the trout _3a- and _3b-AR are closely related because they arose from the genome duplication event that occurred near the base of the salmonid lineage (10, 26, 45).

Tissue-specific expression patterns of the rainbow trout ₃-AR genes were determined using the highly sensitive RPA and gene-specific probes that hybridized to a variable region that included the third intracellular loop. Rainbow trout _3a-AR mRNA was present at high levels in the gill and heart and at lower levels in red muscle (Fig. 3). The presence of rainbow trout _3a-AR mRNA in gill, heart, and red muscle is consistent with previous binding studies on these tissues from trout and other fish that demonstrated the presence of a -AR (11, 15, 18, 40). Rainbow trout _3b-AR is expressed predominantly and at high levels in the RBC (Fig. 3 and 4). This result is in contrast with previous physiology/pharmacology studies that suggested a ₁-AR subtype-controlled -NHE activity in the trout RBC (41). Low levels of _3b-AR mRNA were also detected in other tissues (Fig. 4).

These molecular data clearly demonstrate that the trout RBC contains a _3b-AR rather than a ₁-AR subtype as previously suggested (41). The incongruence between the molecular and physiological data could be explained by the presence of two -AR subtypes in trout blood; however, several lines of evidence argue against multiple -ARs in trout blood. At the pharmacological level, CGP binding to the trout RBC -AR best fit a one-site model, whereas at the molecular level the absence of rainbow trout ₂- and _3a-AR expression in trout blood was clearly demonstrated (Figs. 3 and 4; and Ref. 24). In addition, no potential cross-hybridization products are apparent in either Northern blot (result not shown) or RPA analyses of trout RBC RNA using rainbow trout ₂-, _3a-, or _3b-AR-specific probes (Fig. 3 and Ref. 24). Finally, numerous cloning experiments using different primer sets designed to be either universal to all -AR subtypes or specific to ₁-AR subtypes yielded only _3b-AR sequences from trout blood. A second possible explanation for the incongruence between the molecular and physiological data may be that the previous pharmacological classification of the trout RBC -AR as a ₁-subtype was based on a rank order of potency of NE > Epi for this trout receptor. Unfortunately, this potency order is characteristic of both mammalian ₁-and ₃-AR subtypes (38). Thus the results of these previous studies could actually support the presence of either a ₁- or a ₃-AR subtype in the trout RBC. This possibility led to a more in-depth pharmacological characterization of the rainbow trout RBC _3b-AR.

Competitive displacement of [³H]CGP from RBC _3b-ARs by the nonselective endogenous -AR agonists Epi and NE confirms previous studies and indicates that the trout RBC _3b-AR has a higher affinity for NE than Epi (Fig. 5). However, of the subtype-selective -AR agonists, only clenbuterol (₂ selective) produced significant displacement of [³H]CGP from the _3b-AR (Fig. 5). The use of -AR-subtype-selective antagonists also indicated ₂-AR binding properties, whereas the high affinity of the rainbow trout _3b-AR for the nonselective -AR antagonists propranolol and nadalol (Fig. 6) is not consistent with mammalian ₃-ARs that show low affinities for these same compounds (38). Taken together, these results argue in favor of ₂-AR-like binding characteristics for the trout RBC _3b-AR. However, experiments examining the in vitro ability of various -AR agonists and antagonists to elicit changes in blood pH through stimulation or inhibition of -AR signaling in the trout RBC do not concur with a ₂-AR-like pharmacology for this trout -AR. Among -AR agonists, isoproterenol (-AR nonselective) elicited the largest drop in blood pH followed by BRL (₃ selective), with dobutamine (₁ selective) and clenbuterol (₂ selective) failing to elicit significant decreases in blood pH (Fig. 7). Inhibition of the RBC isoproterenol response by various compounds surprisingly demonstrated that clenbuterol, a ₂-AR agonist, produced the highest level of inhibition (77.8%). Thus clenbuterol acts as an antagonist at the trout RBC _3b-AR. Among -AR antagonists, ICI (₂ selective) produced the largest inhibition of the isoproterenol response, whereas atenolol (₁ selective) and CGP (₁/₂ mixed) failed to significantly inhibit the isoproterenol response (Fig. 7). The results of the competitive displacement and activation/inhibition studies demonstrate that the trout _3b-AR possesses novel pharmacological properties relative to those of the three well-defined mammalian -AR subtypes. Again, these results do not support the previous ₁-AR subtype classification of this receptor that was based on fewer AR ligands. Furthermore, these results are in agreement with a previous finding that -ARs from nonmammalian vertebrates such as amphibians and fish do not fit into the pharmacological categories established for mammalian -ARs (14). This finding suggests that caution is necessary when defining fish AR subtypes using ligands developed for mammalian receptor systems.

This study presents molecular data describing two new members of the trout -AR gene family (rainbow trout _3a- and _3b-AR). Our classification of these trout -ARs as homologs to the mammalian ₃-AR is based on a phylogenetic analysis that received high statistical support. However, the rainbow trout _3b-AR possesses unique pharmacological properties that distinguish this trout receptor from the three pharmacologically defined mammalian -AR subtypes, including the ₃-AR. Despite this fact, the classification of this trout receptor as a _3b-AR reflects its evolutionary relationship to other vertebrate -ARs. The high level of _3b-AR expression in trout blood is strong evidence that this receptor is responsible for controlling RBC -NHE activity. Control of trout RBC -NHE activity represents a novel role for a ₃-AR subtype in fish and demonstrates that the unique characteristics of the components in the trout RBC -AR/-NHE system extend beyond the adrenergically activated Na⁺/H⁺ exchanger.

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 
These studies were supported by grants from the Natural Sciences and Engineering Research Council of Canada to G. Drouin, S. F. Perry, and T. W. Moon.

	ACKNOWLEDGMENTS

 
The authors thank P. Desforge and M. Bayaa for technical assistance with surgical procedures, collection of blood samples, and QPCR experiments.

	FOOTNOTES

 

Address for reprint requests and other correspondence: J. G. Nickerson, Dept. of Biology, Univ. of Ottawa, 150 Louis Pasteur, PO Box 450, Station A Ottawa, Ontario, Canada K1N 6N5.

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.

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION DISCLOSURES REFERENCES

 

1. Atgié C, Tavernier G, D'Allaire F, Bengtsson T, Marti L, Carpéné C, Lafontan M, Bukowiecki LJ, and Langin D. ₃-Adrenoceptor in guinea pig brown and white adipocytes: low expression and lack of function. Am J Physiol Regul Integr Comp Physiol 271: R1729-R1738, 1996.[Abstract/Free Full Text]
2. Borgese F, Malapert M, Fievet B, Pouyssegur J, and Motais R. The cytoplasmic domain of the Na⁺/H⁺ exchangers (NHEs) dictates the nature of the hormonal response: behavior of a chimeric human NHE1/trout beta NHE antiporter. Proc Natl Acad Sci USA 91: 5431-5435, 1994.[Abstract/Free Full Text]
3. Borgese F, Sardet C, Cappadoro M, Pouyssegur J, and Motais R. Cloning and expression of a cAMP-activated Na⁺/H⁺ exchanger: evidence that the cytoplasmic domain mediates hormonal regulation. Proc Natl Acad Sci USA 89: 6765-6769, 1992.[Abstract/Free Full Text]
4. Cantor CR and Smith CL. Genetic analysis. In: Genomics, the Science and Technology Behind the Human Genome Project. Toronto, Canada: Wiley, 1999.
5. Chen XH, Harden TK, and Nicholas RA. Molecular cloning and characterization of a novel -adrenergic receptor. J Biol Chem 269: 24810-24819, 1994.[Abstract/Free Full Text]
6. Devic E, Paquereau L, Steinberg R, Caput D, and Audigier Y. Early expression of a ₁-adrenergic receptor and catecholamines in Xenopus oocytes and embryos. FEBS Lett 417: 184-190, 1997.[ISI][Medline]
7. Dixon RAF, Sigal IS, Candelore MR, Register RB, and Scattergood W. Structural features required for ligand binding to the -adrenergic receptor. EMBO J 6: 3269-3275, 1987.[ISI][Medline]
8. Drouin G. Characterization of the gene conversions between the multigene family members of the yeast genome. J Mol Evol 55: 14-23, 2002.[ISI][Medline]
9. Fabbri E and Moon TW. Adrenergic receptors and second messenger systems in liver of vertebrates. In: Perspectives in Comparative Endocrinology, edited by Davey KG, Peter RE, and Tobe SS. Ottawa, Canada: NRC, 1994, p. 499-506.
10. Ferguson MM and Allendorf FW. Evolution of the fish genome. In: Biochemistry and Molecular Biology of Fishes, edited by Hochachka P and Mommsen T. Amsterdam: Elsevier Science B.V., 1991, vol. 1, p. 25-31, 1991.
11. Gamperl AK, Vijayan MM, Pereira C, and Farrell AP. -Receptors and stress protein 70 expression in hypoxic myocardium of rainbow trout and chinook salmon. Am J Physiol Regul Integr Comp Physiol 274: R428-R436, 1998.[Abstract/Free Full Text]
12. Granneman JG and Lahners K. Analysis of human and rodent ₃-adrenergic receptor messenger ribonucleic acids. Endocrinology 135: 1025-1031, 1994.[Abstract]
13. Guizouarn H, Borgese F, Pellissier B, Garcia Romeu F, and Motais R. Role of protein phosphorylation and dephosphorylation in activation and desensitization of the cAMP-dependant Na⁺/H⁺ antiport. J Biol Chem 268: 8632-8639, 1993.[Abstract/Free Full Text]
14. Jozefowski SJ and Plytycz B. Characterization of -adrenergic receptors in fish and amphibian lymphoid organs. Dev Comp Immunol 22: 587-603, 1998.[ISI][Medline]
15. Keen JE, Vianzon DM, Farrell AP, and Tibbits GF. Thermal acclimation alters both adrenergic sensitivity and adrenoceptor density in cardiac tissue of rainbow trout. J Exp Biol 181: 27-47, 1993.[Abstract]
16. Kompa AR, Gu X, Evans BA, and Summers RJ. Desensitization of cardiac -adrenoceptor signaling with heart failure produced by myocardial infarction in the rat. Evidence for the role of Gi but not Gs or phosphorylating proteins. J Mol Cell Cardiol 31: 1185-1201, 1999.[ISI][Medline]
17. Lomsaney JW, Allen LF, and Lefkowitz RJ. Cloning and regulation of catecholamine receptor genes. In: Molecular Endocrinology: Basic Concepts and Clinical Correlations, edited by Weintraub BD. New York: Raven, 1995.
18. Lortie M and Moon TW. The rainbow trout skeletal muscle -adrenoceptor system: characterization and signaling. Am J Physiol Regul Integr Comp Physiol 284: R689-R697, 2003.[Abstract/Free Full Text]
19. Malapert M, Guizouarn H, Fievet B, Jahns R, Garcia Romeu F, Motais R, and Borgese F. Regulation of Na⁺/H⁺ antiporter in trout red blood cells. J Exp Biol 200: 353-360, 1997.[Abstract]
20. Mayor F Jr, Penela P, and Ruiz-Gomez A. Role of G-protein coupled receptor kinase 2 and arrestins in -adrenergic receptor internalization. Trends Cardiovasc Med 8: 234-240, 1998.[ISI][Medline]
21. McNeel RL and Mersmann H. Distribution and quantification of beta₁-, beta₂-, and beta₃-adrenergic receptor subtype transcripts in porcine tissues. J Anim Sci 77: 611-621, 1999.[Abstract/Free Full Text]
22. Motais R, Borgese F, Fievet B, and Garcia Romeu F. Regulation of Na⁺/H⁺ exchange and pH in erythrocytes of fish. Comp Biochem Physiol 102: 597-602, 1992.[Medline]
23. Muzzin P, Revelli JP, Kuhne F, Gocayne JD, McCombie WR, Venter JC, Giacobino JP, and Fraser CM. An adipose tissue specific -adrenergic receptor. J Biol Chem 266: 24053-24058, 1991.[Abstract/Free Full Text]
24. Nickerson JG, Dugan SG, Drouin G, and Moon TW. A putative ₂-adrenoceptor from the rainbow trout (Oncorhynchus mykiss). Eur J Biochem 268: 6465-6472, 2001.[ISI][Medline]
25. Nikinmaa M. Membrane transport and control of hemoglobin-oxygen affinity in nucleated erythrocytes. Physiol Rev 72: 301-321, 1992.[Free Full Text]
26. Ohno S. Evolution by Gene Duplication. New York: Springer-Verlag, 1970.
27. Perry SF and McDonald G. Gas exchange. In: The Physiology of Fishes. Boca Raton, FL: CRC, 1993, p. 251-277.
28. Perry SF, Reid SG, and Salama A. The effect of repeated physical stress on the -adrenergic response of the rainbow trout red blood cell. J Exp Biol 199: 549-562, 1996.[Abstract]
29. Perry SF and Thomas S. Rapid respiratory changes in trout red blood cells during sodium/proton exchange activation. J Exp Biol 180: 27-37, 1993.[Abstract]
30. Pfaffl WM. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29: 2002-2007, 2001.
31. Randall DJ and Perry SF. Catecholamines. In: Fish Physiology, edited by Hoar W, Randall DJ and Farrell AP. San Diego: Academic, 1992, vol. XIIB, p. 255-300.
32. Reid SD, LeBras YM, and Perry SF. The in vitro effects of hypoxia on the trout (Oncorhynchus mykiss) erythrocyte -adrenergic signal transduction system. J Exp Biol 176: 103-116, 1993.[Abstract]
33. Reid SG, Bernier NJ, and Perry SF. The adrenergic stress response in fish: control of catecholamine storage and release. Comp Biochem Physiol 120C: 1-27, 1998.
34. Rodriguez-Cuenca S, Pujol E, Justo R, Frontera M, Oliver J, Gianotti M, and Roca P. Sex-dependent thermogenesis, differences in mitochondrial morphology and function, and adrenergic response in brown adipose tissue. J Biol Chem 277: 42958-42963, 2002.[Abstract/Free Full Text]
35. Sawyer SA. GENECONV: A Computer Package for the Statistical Detection of Gene Conversion. Department of Mathematics, Washington University in St. Louis, MO (http://www.math.wustl.edu/~sawyer), 1999.
36. Soivio A, Nynolm K, and Westman K. A technique for repeated sampling of the blood of individual resting fish. J Exp Biol 63: 207-217, 1975.[Abstract/Free Full Text]
37. Strosberg AD. Structure, function and regulation of adrenergic receptors. Protein Sci 2: 1198-1209, 1993.[Abstract]
38. Strosberg AD. Structure and function of the ₃-adrenergic receptor. Annu Rev Pharmacol Toxicol 37: 421-450, 1997.[Medline]
39. Strimmer K and von Haeseler A. Quartet puzzling: a quartet maximum likelihood method for reconstructing tree topologies. Mol Biol Evol 13: 964-969, 1996.[ISI]
40. Sundin LI. Responses of the branchial circulation to hypoxia in the Atlantic cod, Gadus morhua. Am J Physiol Regul Integr Comp Physiol 268: R771-R778, 1995.[Abstract/Free Full Text]
41. Tetens V, Lykkeboe G, and Christensen NJ. Potency of adrenaline and noradrenaline for beta-adrenergic proton extrusion from red blood cells of rainbow trout, Salmo gairdneri. J Exp Biol 134: 267-280, 1988.[Abstract/Free Full Text]
42. Thompson JD, Higgins DG, and Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673-4680, 1994.[Abstract/Free Full Text]
43. Valdenaire O and Vernier P. G protein coupled receptors as modules of interacting proteins: a family meeting. Prog Drug Res 49: 174-218, 1997.
44. Wolf K. Physiological salines for freshwater teleosts. Progr Fish Cult 25: 135-140, 1963.
45. Wolfe HK. Yesterday's polyploids and the mystery of diploidization. Nature Rev Gene 2: 333-341, 2001.[ISI][Medline]
46. Yarden Y, Rodriguez H, Wong SKF, Brandt DR, May DC, Burnier J, Harkins RN, Chen EY, Ramachandran J, Ull-rich A, and Ross EM. The avian beta-adrenergic receptor: primary structure and membrane topology. Proc Natl Acad Sci USA 83: 6795-6799, 1986.[Abstract/Free Full Text]

This article has been cited by other articles:

				C. L. Brett, M. Donowitz, and R. Rao Evolutionary origins of eukaryotic sodium/proton exchangers Am J Physiol Cell Physiol, February 1, 2005; 288(2): C223 - C239. [Abstract] [Full Text] [PDF]

				M. Nikinmaa {beta}3-Adrenergic receptors--studies on rainbow trout reveal ancient evolutionary origins and functions distinct from the thermogenic response Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2003; 285(3): R515 - R516. [Full Text] [PDF]

This Article