Abstract
Angiotensinogen belongs to family A serine protease inhibitors (SERPIN family) and we have cloned and characterized SERPIN genes in two lamprey species, which possess all the properties of angiotensinogen. The putative angiotensinogens in lampreys can be considered as an evolutionary link between SERPIN and other angiotensinogen according to the phylogenetic analyses. The inferred sea lamprey angiotensinogen gene was expressed abundantly in liver and to a lesser extent in other tissues. The predicted lamprey angiotensin I (Ang I) sequence was unique and different from the teleost-type Ang I identified previously by the incubation of lamprey plasma with its kidney extract. Therefore, we characterized and compared the biochemical and physiological properties of this native lamprey Ang II (LpAng II) (EEDYDERPYMQPF) with teleost-type Ang II (NRVYVHPF). Using a newly developed RIA for LpAng II, plasma levels in Japanese lamprey were measured (157.4±35.2 fmol/ml, n=6), but teleost-type Ang II was undetectable. In conscious cannulated lamprey, LpAng II at 100 pmol/kg elicited a transient vasodepressor effect. At doses higher than 300 pmol/kg, a biphasic cardiovascular response with an initial vasodepressor effect followed by a transient rebound vasopressor effect was observed in a dose-dependent manner. However, teleost-type Ang II was not vasoactive up to 1 nmol/kg. In Japanese eel, LpAng II injection up to 3 nmol/kg did not alter the cardiovascular parameters. Our results suggested that the renin–angiotensin system first appeared in cyclostomes, and LpAng II could be important for the regulation of cardiovascular dynamics in lampreys because of its potent and acute vasoactive effect.
Introduction
The renin–angiotensin system (RAS) is important for the regulation of salt and water balance in vertebrates ( Olson 1992, Kobayashi & Takei 1996). Angiotensinogen, the precursor protein of the peptide cascade system, is highly variable among vertebrates, but reasonable homology is observed in angiotensin I (Ang I) sequences located at the N-terminus ( Watanabe et al. 2009). The presence of the RAS in cyclostomes has been the subject of a long debate ( Nishimura et al. 1970, Nishimura 1985). Several studies have shown that RAS components including Ang I, Ang II, and angiotensin-converting enzyme-like (ACE-like) activity were present in river lamprey (Lampetra fluviatilis) and sea lamprey (Petromyzon marinus; Rankin et al. 2001, 2004, Cobb et al. 2002, Takei et al. 2004). However, the majority of ACE-like activity was found in the brain of river lamprey ( Cobb et al. 2002) instead of the gills as in teleost fishes ( Olson 1992). Furthermore, [Asn1, Val5, Thr9]-Ang I, which is similar to the Ang Is of other teleosts, was isolated from the incubation of plasma (angiotensinogen source) and kidney extract (renin source) in the river lamprey ( Rankin et al. 2004) and sea lamprey ( Takei et al. 2004). However, vasopressor effect of [Asn1, Val5]-Ang II was extremely low in river lampreys ( Rankin et al. 2004), and i.p. injection of Ang II (163 nmol/kg) or papaverine, a stimulant of endogenous RAS in various vertebrate species ( Tierney et al. 1995a), failed to stimulate drinking behavior ( Rankin et al. 2001). Therefore, it is still not clear whether [Asn1, Val5]-Ang II exerts any physiologically relevant effect in lampreys.
To date, molecular data on the RAS in lamprey are limited. In the sea lamprey genome, we have identified a putative angiotensinogen gene that belongs to family A serine protease inhibitor (SERPIN family), and the putative protein possesses an Ang I that has a sequence that is different from [Asn1, Val5, Thr9]-Ang I. The discrepant Ang I sequences from biochemical and molecular data have prompted us to embark on the present investigation. In the present study, we aim to provide evidence to show that the genome-identified angiotensinogen gives rise to a native functional angiotensin in lamprey. Specific RIA was developed to measure the plasma level of the newly identified lamprey Ang II (LpAng II). Cardiovascular functions of LpAng II were examined in conscious lamprey. The present result provides important insights on the evolutionary origin of the RAS and its functional role in vertebrates.
Materials and Methods
cDNA cloning and genome survey of angiotensinogens in vertebrates
Initially, a putative angiotensinogen gene/SERPIN was identified in Sea Lamprey Genome database (contig1818). The contig sequence was subjected to analysis by GENESCAN program to identify putative cDNA. Sea lamprey (P. marinus) and Japanese lamprey (Lethenteron japonicum) tissues and cDNA were obtained from previous collections of our laboratory ( Wong & Takei 2009). Specific primers (SL_AGT_F1: 5′-CTACATGCAGCCCTTCCATCTAATTC-3′ and SL_AGT_R1 5′-GAAGATCACAGCTCTCGTTTTCTCATC-3′) were designed from putative coding regions to amplify partial fragments of angiotensinogen in sea lamprey and Japanese lamprey. To clone the full-length cDNA of angiotensinogen in both lamprey species using rapid amplification of cDNA ends, liver cDNA was prepared in each case using a SMART cDNA Library Construction kit (Clontech Laboratories) according to the manufacturer's protocol. PCR was performed using an ABI 9700 thermal cycler (Applied Biosystems, Foster City, CA, USA) with Takara ExTaq DNA Polymerase reagents (Takara Bio, Inc., Otsu, Shiga, Japan). PCR products were electrophoresed on 1.2% agarose gels stained by ethidium bromide, excised, extracted using an UltraClean 15 DNA Purification kit (Mo Bio Laboratories, Inc., Carlsbad, CA, USA), cloned into pT7blue vector (Novagen, Madison, WI, USA), and sequenced using a BigDye Terminator Cycle sequencing kit and an ABI 3130 DNA sequencer (Applied Biosystems). The full-length angiotensinogen sequence of sea lamprey was aligned with the genomic contig1818 to resolve the exon–intron structure.
Tissue distribution of angiotensinogen mRNA
Total RNA samples were obtained from various tissues including brain, gill, atrium, ventricle, liver, anterior kidney, posterior kidney, anterior intestine, posterior intestine, skeletal muscle, and skin using Isogen RNA extraction system (Nippongene, Toyama, Japan). RNA samples were reverse-transcribed to cDNA using SuperScript III First-Strand Synthesis for RT-PCR (Invitrogen) according to the manufacturer's protocol. Specific primers were designed spanning across introns to prevent amplification of genomic DNA (SL_AGT_Exp_F: 5′-GTCAATGTCATTTATTTCAAAGGATCGTG-3′ and SL_AGT_Exp_R: 5′-TATGAGTGATCTTGTCCACGTGTAGCTT-3′). PCR was performed using an ABI 9700 thermal cycler (Applied Biosystems) with Takara ExTaq DNA polymerase reagents (Takara Bio, Inc.), and PCR products were electrophoresed on 1.2% agarose gel stained by ethidium bromide.
Phylogenetic analysis and mature peptide prediction
Extensive genome search was performed to collect most of the available angiotensinogen or putative angiotensinogen sequences from each species under genome project coverage. Angiotensinogen sequences that are available on the GenBank nucleotide and expressed sequence tag (EST) databases were also collected for phylogenetic reconstruction. Full-length amino acid sequences of various species were aligned using Mega version 4, and a phylogenetic tree was constructed using the neighbor joining method (JTT) and maximum parsimony ( Tamura et al. 2007). A bootstrap test was performed with 1000 replicates to validate the phylogenetic relationships. To further validate the phylogenetic relationship, the same alignment was analyzed by Bayesian method ( Ronquist & Huelsenbeck 2003) to generate an alternative phylogenetic tree for comparison. The signal peptides of angiotensinogens in P. marinus and L. japonicum were predicted using neural networks and hidden Markov model. Homologous regions of vertebrate Ang II and LpAng II were aligned to identify putative Ang II in lampreys.
Antibody production for LpAng II
LpAng II (EEDYDERPYMQPF) was synthesized (Peptide Institute, Inc., Ibaraki, Osaka, Japan) according to the predicted sequence of Japanese lamprey. LpAng II was N-terminal conjugated to keyhole limpet hemocyanin (KLH) using glutaraldehyde as a catalyst, and the protein complex was used in immunization in rabbits to produce polyclonal antibodies. The rabbits were first immunized with Freud's complete adjuvant followed by two booster injections with Freud's incomplete adjuvant. The interval between each injection was 3 weeks. Titers of antibodies were monitored after each bleed by enzyme immunoassay. The rabbits were killed for final bleed after the third immunization.
Development of a RIA for LpAng II
LpAng II was radiolabeled by 125I using the lactoperoxidase method ( Marchalonis 1969). 125I-LpAng II was diluted in RIA buffer containing 0.1% RIA grade BSA to produce 10 000 c.p.m. per assay tube. The titers of the antibodies were determined by serial dilution, and maximum binding was adjusted at 20% of the total binding. Incubation of standard peptides and unknown samples with radiolabeled tracer was carried out at 4 °C for 24 h. Separation of free and bound fractions was achieved by precipitation using a secondary antibody, goat anti-rabbit IgG (Sigma), and 16% polyethylene glycol followed by centrifugation at 3000 g for 1 h at 25 °C and then 30 min at 4 °C. Unbound fractions were removed by aspiration, and specific binding in the pellet was measured using a gamma counter (Perkin Elmer, Waltham, MA, USA). A standard curve ranging from 10 to 100 000 fmol/ml was constructed. Cooperativity of antibody binding was observed at 10–300 fmol/ml region, resulting in a specific binding over maximum binding. Therefore, unknown samples were serial diluted during measurement to delineate between specific displacement and no displacement. Specificity of the LpAng II RIA was verified by measuring the cross reactivity of LpAng II antibody to [Asn1, Val5]-Ang II and [Asn1, Pro3, Ile5]-Ang II.
Qualitative and quantitative analyses of different angiotensins in lamprey plasma
Upstream migrating adult Japanese lampreys were obtained from local fishermen of Niigata prefecture. All upstream migrating lampreys have regressed intestines and full-grown gonads. They were kept in a freshwater recirculating system maintained at 10 °C. All animal experimentation procedures were approved by the Animal Experiment Committees of the University of Tokyo and performed in accordance with the Manual of Animal Experiments prepared by the Committee. Lampreys were anesthetized using 0.1% ethyl 3-aminobenzoate methanesulfonate (Sigma) neutralized by sodium bicarbonate until ventilation movement ceased. Blood samples were withdrawn by heart puncture into syringes containing an inhibitor cocktail (0.05 M 1,10-phenanthroline, 0.225 M potassium EDTA, and 0.1 TIU aprotinin) that inhibits peptide degradation. Blood samples were centrifuged immediately at 8000 g for 5 min, and plasma fractions were frozen at −20 °C until use.
Plasma samples were extracted by equal volumes of acidic acetone (acetone:water:1 M HCl=40:5:1; Brown et al. 2005). The partially purified plasma was further resolved by reverse-phase HPLC utilizing an analytical column (ODS 100 V column, 5 μm, 4.6 mm I.D.×25.0 cm, Tosoh, Tokyo, Japan). A linear gradient from 15 to 35% acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min over 40 min was used for separation, and a single peptide sample could be eluted within 0.4 min under this profile. HPLC fractions were collected at 1 min intervals and dried by lyophilization. The HPLC-resolved fractions were reconstituted in RIA buffer for the measurement of [Asn1, Val5]-Ang II ( Tsuchida & Takei 1998) and LpAng II using specific RIAs for each peptide. All measurements were performed in duplicate.
Effects of LpAng II on ventral aortic pressure in lamprey and eel
Japanese lampreys were anesthetized as described above. The ventral aorta was cannulated (SP31 polyethylene tubing, I.D. 0.5 mm, O.D. 0.8 mm, Natsume, Tokyo, Japan) using a procedure modified from the cannulation of eel ( Tsuchida & Takei 1998). After operation, the lampreys were allowed to recover from surgical stress in an elongated plastic box with constant supply of aerated freshwater at 10 °C for 24 h before injections.
The ventral aortic cannula was filled with heparinized saline (200 U/ml, 0.9% NaCl) and connected to a DX-300 pressure transducer (Nihon Koden, Tokyo, Japan) coupled to a monitoring system (MTS00658, Medical Try System, Tokyo, Japan) via a three-way stopcock for the measurement of blood pressure and heart rate. The same cannula was used for drug administration and blood pressure measurement. Cultured Japanese eels were cannulated for measurement of ventral aortic pressure using the same procedure as for lamprey. LpAng II and [Asn1, Val5]-Ang II were diluted in saline (0.9% NaCl), and bolus injections containing various doses of the peptides were administered in 50 μl followed by a flush of 50 μl saline.
Statistical analysis
Changes in average blood pressure and pulse pressure of the lamprey and eel after injections were analyzed by two-way ANOVA followed by Bonferroni test.
Results
Cloning, tissue expression, and phylogenetic analysis of lamprey angiotensinogen
The alignment of sea lamprey and Japanese lamprey angiotensinogen is shown in Fig. 1. Both amino acid sequences showed typical angiotensinogen characteristics, including a putative Ang I sequence after the signal peptide, glycosylation sites for addition of sugar chain, and a SERPIN-specific motif at the C-terminal region. The amino acid identities of sea lamprey angiotensinogen to those of elephant fish (Callorhinchus milii) and zebrafish (Danio rerio) were only 24 and 26% respectively. However, the putative Ang I sequences of both lamprey species showed a moderate homology to other vertebrate representatives at the amino acid and nucleotide levels ( Fig. 2). Among elasmobranchs and lamprey, a codon usage bias was observed in the nucleotide region encoding [Arg2] and [Pro3] of elasmobranch Ang Is. Despite high degeneracy of codon usage in [Arg] and [Pro], identical nucleotide sequence was found between lamprey and elasmobranchs. Although considerable amino acid substitutions are present among the Ang I sequences, the nucleotide alignment showed that all the corresponding substitutions can be attributed to single base changes. The gene structure of lamprey angiotensinogen is highly comparable to that of other vertebrate representatives from fish to mammal ( Fig. 3), including those of chondrichthyans ( Watanabe et al. 2009). Lamprey angiotensinogen was expressed in various tissues including brain, atrium, kidney, intestine, and muscle, and its expression in liver was relatively higher than other tissues ( Fig. 4). The phylogenetic tree ( Fig. 5) showed that lamprey angiotensinogens were closely related to SERPIN A family and chondrichthyan angiotensinogens. The phylogenetic trees inferred by neighbor joining, maximum parsimony, and Bayesian methods had the same topology, and therefore only the former was shown.
Amino acid sequence comparison between the angiotensinogens of sea lamprey (Petromyzon marinus) and Japanese lamprey (Lethenteron japonicum). Putative lamprey Ang I sequences are boxed. Predicted signal peptide cleavage site is indicated by an arrow. Putative N-glycosylation sites are underlined, and the shaded region represents feature motif of SERPIN.
Citation: Journal of Endocrinology 209, 1; 10.1530/JOE-10-0422
Alignments of several Ang Is of different vertebrate representatives at the amino acid and nucleotide levels. Difference in corresponding amino acid sequence in lamprey Ang I can be attributed to the single-base changes at the nucleotide level. Shaded regions in amino acid and nucleotide alignment indicate a conserved codon usage among lamprey and chondrichthyan species.
Citation: Journal of Endocrinology 209, 1; 10.1530/JOE-10-0422
Exon–intron structures of angiotensinogens (AGT) of sea lamprey and other vertebrate representatives. The gene structure of lamprey was reconstructed by aligning cloned cDNA against the genomic contig. Angiotensinogen gene structures of Homo sapiens (NG_008836), Gallus gallus (XM_419584), Anolis carolinensis (Scaffold_200), Xenopus tropicalis (Scaffold_JG14), and Danio rerio (BC095585) were obtained or predicted from GenBank or Ensembl genome browser. Putative lamprey angiotensin I (LpAng I) and other vertebrate Ang I, indicated by the filled boxes, are located at the homologous position of exon 1.
Citation: Journal of Endocrinology 209, 1; 10.1530/JOE-10-0422
Distribution of angiotensinogen (AGT) mRNAs in various tissues of sea lamprey. GAPDH was used for internal control. B, brain; G, gill; A, atrium; V, ventricle; L, liver; K, kidney; AK, anterior kidney; AI, anterior intestine; PI, posterior intestine; T, testis; M, skeletal muscle and SK, skin.
Citation: Journal of Endocrinology 209, 1; 10.1530/JOE-10-0422
Phylogenetic tree of vertebrate angiotensinogen depicted by the neighbor joining method. Numbers on the branches are the bootstrap values of 1000 replicates. Scale bar represents 20% amino acid substitution. Danio SERPIN A was used as an outgroup. GenBank accession numbers or genome contig numbers are as follows: Tursiops (Scaffold_106652), Bos (NM_001114082), Canis (XM_536348), Myotis (Scaffold_12156), Equus (XM_001496084), Oryctolagus (GeneScaffold_2959), Rattus (BC078741), Cavia (Scaffold_16), Mecaca (Chromosome 1), Pongo (Chromosome 1), Homo (NM_000029), Gorilla (AF188488), Echinops (GeneScaffold_3833), Monodelphis (Chromosome 2), Xenopus (Scaffold_JG14), Anolis (Scaffold_200), Gallus (XM_419584), Meleagris (Chromosome 2), Taeniopygia (Chromosome 3), Anas (Scaffold759), Danio (BC095585), Takifugu (BK001021), Plecoglossus (AM921805), Salmo (NM_001140446), Oryzias (Chromosome 15), Rhabdosargus (DQ136311), Gasterosteus (GroupVI), Callorhinchus (AB433535), Triakis (AB433533), Dasyatis (AB433534), Leucoraja (AB433536), Petromyzon (GU566000), Lethenteron (GU391575), Lamptera (FM954978), and Danio SERPIN A (BC075902).
Citation: Journal of Endocrinology 209, 1; 10.1530/JOE-10-0422
Characterization of different Ang II forms in lamprey plasma
The polyclonal antibodies raised against LpAng II exhibit positive cooperativity binding as shown in the standard curve ( Fig. 6). The specific binding, ranging from 10 to 300 fmol/ml, was slightly higher than that of maximum binding. In practice, unknown samples were serially diluted for measurement to validate specific bindings. The LpAng II assay is highly specific, with only 0.14 and 0.53% cross reactivities to teleost [Asn1, Val5]-Ang II and elasmobranch [Asn1, Pro3, Ile5]-Ang II respectively. Intra-assay and inter-assay coefficients of variations were 4.4 and 12.3% respectively (n=8). The LpAng II assay has a detection range of 10–3000 fmol/ml.
A standard curve of the RIA of lamprey Ang II (LpAng II). The RIA exhibits cooperativity characteristics resulting in increased specific binding at particular concentration. Serially diluted lamprey plasma samples pooled from three individuals show parallelism to the standard curve. Upper scale shows the dilution factors of the pooled plasma sample.
Citation: Journal of Endocrinology 209, 1; 10.1530/JOE-10-0422
A pooled plasma sample from three individuals was shown to exhibit positive cooperative characteristics, and the curve derived from serial diluted plasma was parallel to the standard curve of LpAng II ( Fig. 6). Average plasma levels of LpAng II were 157.4±35.2 fmol/ml (n=6), but [Asn1, Val5]-Ang II was undetectable in the same plasma samples ( Fig. 7). In the same sets of plasma HPLC fractions, immunoreactive LpAng II was detected at the elution position identical to that of synthetic standard. The LpAng II RIA did not detect any extra peaks in the HPLC-resolved plasma fractions, indicating a high specificity of the developed assay as well as adequate protection of sample from peptide degradation.
Immunoreactive Ang II and LpAng II in lamprey plasma measured by respective RIAs after reverse-phase HPLC fractionation. Plasma samples from six individuals were examined. Arrows indicate the elution positions of synthetic [Asn1, Val5]-Ang II, [Asn1, Ile5]-Ang II, and LpAng II.
Citation: Journal of Endocrinology 209, 1; 10.1530/JOE-10-0422
Cardiovascular effects of different Ang II forms in lamprey and eel
The average ventral aortic pressure of Japanese lamprey was 19.4±1.3 mmHg, which was similar to the reported values in other lamprey species ( Donald 1998, Farrel 2007). LpAng II elicited a dose-dependent, biphasic response in ventral aortic pressure in Japanese lamprey ( Fig. 8). Bolus injection of LpAng II induced a transient decrease in blood pressure followed by a rebound increase in blood pressure. The original blood pressure tracing showed that the initial vasodepressor response occurred within 1–2 min, and the rebound increase started 2–3 min after the injection. Furthermore, the vasodepressor effect was more potent, as significant pressure drop was observed with a dose of 100 pmol/kg but the rebound increase was only significant with higher doses starting from 300 pmol/kg when compared with the resting levels. Despite a prominent change in blood pressure, the heart rate was not altered during the biphasic response. After the rebound increase in blood pressure, there was a significant elevation in the pulse pressure at doses starting from 300 pmol/kg. We did not include the cardiovascular data of doses higher than 1000 pmol/kg because temporal cardiac arrest was sometimes observed, and then the lamprey started undulating violently within the plastic box when higher doses of LpAng II were administered. On the other hand, [Asn1, Val5]-Ang II did not change aortic pressure of lamprey at a series of doses up to 1 nmol/kg ( Fig. 9).
Dose-related effect of bolus injection of LpAng II on ventral aortic pressure (BP; B), pulse pressure (PP; C), and heart rate (HR; D) in Japanese lamprey. A shows an example of the original tracing of ventral aortic pressure and heart rate recording. Numbers in parentheses indicate the number of observations in each treatment. Values are shown as mean±s.e.m. Significant differences are denoted by asterisks (two-way ANOVA, Bonferroni test, ***P<0.001).
Citation: Journal of Endocrinology 209, 1; 10.1530/JOE-10-0422
Dose-related effect of bolus injection of [Asn1, Val5]-Ang II on ventral aortic pressure (BP; B) and pulse pressure (PP; C) in Japanese lamprey. A shows an example of the original tracing of ventral aortic pressure and heart rate (HR) recording. Numbers in parentheses indicate the number of observations in each treatment. Values are shown in mean±s.e.m.
Citation: Journal of Endocrinology 209, 1; 10.1530/JOE-10-0422
To show the species specificity of LpAng II, Japanese eel was used to study the possible effects of LpAng II in teleost. In cannulated eel, LpAng II did not produce any vasoactive response at all doses up to 3 nmol/kg ( Fig. 10). Homologous [Asn1, Val5]-Ang II injection in eels at 1000 pmol/kg induced a profound increase in ventral aortic pressure, serving as a positive control for the responsiveness.
Dose-related effect of bolus injection of LpAng II on ventral aortic pressure (BP) in Japanese eel. Homologous [Asn1, Val5]-Ang II injection was used as a positive control to confirm the responsiveness. Upper panel shows examples of the original tracing of ventral aortic pressure and heart rate (HR) recording. Numbers in parentheses indicate the number of observations in each treatment. Values are shown in mean±s.e.m. Significant differences are denoted by asterisks (two-way ANOVA, Bonferroni test, ***P<0.001).
Citation: Journal of Endocrinology 209, 1; 10.1530/JOE-10-0422
Discussion
In previous studies to demonstrate the presence of RAS in lamprey, plasma and kidney extract of river and sea lamprey were incubated, and using an eel vasopressor bioassay, [Asn1, Val5, Thr9]-Ang I was isolated and identified as the homologous Ang I in lampreys ( Rankin et al. 2004, Takei et al. 2004). However, we recently cloned the putative angiotensinogen genes in sea lamprey and Japanese lamprey respectively according to the genome information. Angiotensinogen belongs to the member 8 of SERPIN family A, and the putative lamprey angiotensinogen possesses a SERPIN-specific motif at the C-terminal region as well as three putative N-glycosylation sites, which are common characteristics of angiotensinogen in vertebrates ( Irving et al. 2007). In addition, the exon–intron structure of the lamprey angiotensinogen was highly comparable to that of other vertebrate species. The putative LpAng I sequences exhibit moderate homology to those of other Ang Is ( Fig. 1), but the sequences were different from that previously isolated using biochemical method. The codon usage bias for [Arg2] and [Pro3] in elasmobranch Ang Is was also observed in lamprey, indicating that the Ang Is of these lineages may have high evolutionary constraint in such motif. Phylogenetic analysis further revealed that the lamprey angiotensinogens were closely related to SERPIN A family, but to a lesser extent to the angiotensinogens of the chondrichthyan lineage. Although the position of lamprey angiotensinogens in the phylogeny was closer to SERPIN A than other angiotensinogens, the lamprey angiotensinogens could be considered as an evolutionary link between the two sister groups, suggesting that lamprey may possess the earliest angiotensinogen in extant vertebrates. It would be interesting to examine whether hagfish possess similar angiotensinogen to that of lamprey.
Analysis of tissue distribution of lamprey angiotensinogen mRNA revealed its expression in various types of tissues including brain, heart, liver, kidney, intestine, and skeletal muscle, with relatively higher expression in the liver. The angiotensinogen expression pattern in lamprey was similar to that of other fish species such as seabream ( Wong et al. 2007), ayu ( Chen et al. 2008), and dogfish ( Watanabe et al. 2009). The liver is the major site of angiotensinogen production in most vertebrate species, though other tissues such as kidney and brain also produce angiotensinogen as a local RAS for supplying Ang II in a paracrine manner ( Paul et al. 2006). The widespread distribution of angiotensinogen gene expression, as observed in lamprey and other fishes, implies that circulating angiotensinogen could be generated by various organs in early diverged vertebrates and that the liver subsequently became the major organ for angiotensinogen production.
In the present study, the RIA developed for LpAng II exhibited cooperativity characteristics. As shown in Fig. 6, the specific bindings of samples and standards were up to 20% higher than those of maximum binding (B0) ranged between 10 and 300 fmol/ml, which is a rare but well-documented phenomenon called cooperative immunoassay ( Ehrlich et al. 1982). In cooperative RIA, the specific binding may exceed the maximum binding because of the formation of an ultra-stable antigen–antibody complex that probably exists as a tetramer or circular complex ( Moyle et al. 1983a, b). Cooperativity is commonly observed when two or more monoclonal antibodies, which possess binding sites for different epitopes that are close to each other on the antigen, are mixed in the immunoassay ( Ehrlich et al. 1982). It is uncommon for a polyclonal antibody to exhibit cooperativity. LpAng II has two consecutive hydrophilic amino acid sequences, [Glu1-Glu2-Asp3] and [Asp5-Glu6-Arg7], which are separated by a [Tyr4] residue. The two hydrophilic groups are predicted to be highly antigenic ( Hopp & Woods 1981); thus, antibodies may recognize these epitopes separately. Because of the closely linked antigenic epitopes in LpAng II, the polyclonal antibodies may possess cooperative characteristics similar to those of a monoclonal antibody mixture ( Ehrlich & Moyle 1984). Furthermore, cooperativity increased the specificity and sensitivity of the RIA, as non-specific bindings are less likely to possess such cooperativity characteristics ( Cheong et al. 1990). The RIA developed for LpAng II was highly specific, as the cross reactivities to other fish-type Ang IIs were low (<1%). Using the newly developed RIA, plasma LpAng II concentration measured in Japanese lamprey (157.4±35.2 fmol/ml) is similar to the Ang II levels reported in other fishes ( Tierney et al. 1995b, Tsuchida & Takei 1998).
It was previously found that [Asn1, Val5]-Ang II could be circulating in river lamprey at a range between 200 and 1000 fmol/ml ( Brown et al. 2005). In the present study, however, [Asn1, Val5]-Ang II was not detected by our RIA system (detection limit 5 fmol/ml) in the plasma of Japanese lamprey caught during upstream migration ( Fig. 7). Here, we hypothesize that the [Asn1, Val5]-Ang II found in lamprey plasma is not the native lamprey form, but could be related to a blood-feeding diet, as mature lampreys are ectoparasites of teleosts. The Japanese lamprey ceases feeding, and the intestine has regressed before the upstream migration begins, but the river lamprey used in previous studies was reported to have a functional intestine capable of acclimation to half-strength seawater ( Brown et al. 2005). We have performed a parallel study to show that teleost-type angiotensin components were present in the buccal gland secretion of the lamprey, which could be a possible source of teleost-type angiotensin (MKS Wong, SA Sower, D Berlinsky & Y Takei, in preparation).
To further investigate the inconsistency between LpAng II and [Asn1, Val5]-Ang II, the vasoactive properties of these peptides in Japanese lamprey and eel were examined. LpAng II produced a dose-dependent, biphasic response in the ventral aortic pressure of lamprey. Upon the bolus injection of LpAng II, ventral aortic blood pressure decreased immediately, followed by a rebound increase in blood pressure. An increase in pulse pressure was observed during the rebound phase, which could be due to an increase in cardiac output and/or systemic resistance. The biphasic response resembled the action of homologous Ang II in fowl ( Nishimura et al. 1982) but not those of other mammals and fishes ( Khosla 1985). It was shown that the vasodepressor action of Ang II in bird was endothelium-dependent, nitric oxide-independent, and signaling through prostaglandin ( Nakamura et al. 1982). The vasopressor effect of Ang II in fowl is mediated through the α-adrenergic pathway and is independent of the vasodepressor effect ( Nishimura et al. 1994). However, information on lamprey cardiovascular dynamics and regulation is limited ( Farrel 2007). The blood vessels in fish are rarely innervated, and the autonomic nervous system is poorly developed ( Donald 1998). The subendothelial layer in lamprey cardiocytes is not innervated but contains catecholamine granules, resulting in a humoral basis of adrenergic stimulation in lamprey ( Augustinsson et al. 1956, Falck et al. 1966). Vagal stimulation in lamprey accelerates heart rate through nicotinic receptors, but cardiac vagal stimulation typically induces a decrease in heart rate through muscarinic receptors in other vertebrates. Stimulation of β-adrenergic receptors also increased heart rate and pulse pressure in lamprey ( Lignon 1979) via the modulation of intracellular calcium availability ( Shiels et al. 2002, Vornanen et al. 2002). In isolated lamprey aortic ring constricted by endothelin, and prostaglandin but not nitric oxide produced significant vasodilation ( Evans & Harrie 2001). Based on available information, direct comparison between the angiotensin effect in bird and lamprey is inappropriate because of the intrinsic differences of their cardiovascular systems. Unlike the Ang II-induced noradrenaline effect in bird, the rebound increase could be a secondary response to the acute vasodepressor effect. Upon injection of LpAng II at doses higher than 1000 pmol/kg, lampreys started undulation movements, which could be a response to increase the venous return to restore the drop in blood pressure. The restoration of blood pressure is unlikely related to β-adrenergic stimulation, as the heart rate remained unchanged. To date, the cardiovascular dynamics in lamprey is not well understood, possibly due to the lack of homologous hormones. We demonstrated that LpAng II could be a potent cardiovascular regulator, but further work is required to investigate its signaling pathways and physiological functions. Isolated blood vessel studies should be performed to elucidate the signaling pathways of LpAng II. Furthermore, possible involvement of LpAng II in kidney functions should be investigated, as Ang II is highly involved in the regulation of glomerular and tubular functions in other vertebrates ( Cobb et al. 1999).
It was previously shown that [Asn1, Val5]-Ang II was a vasopressor in river lamprey, but the minimum effective dose was extraordinarily high (100 000 pmol/kg) and [Asn1, Val5, Thr9]-Ang I was without effect even at the same dose ( Rankin et al. 2004). The minimum effective dose of LpAng II in Japanese lamprey is 3 orders smaller than that of [Asn1, Val5]-Ang II in river lamprey. In the present study, various doses of [Asn1, Val5]-Ang II up to 1 nmol/kg did not produce any observable changes in the blood pressure of Japanese lamprey. Combining the observation that [Asn1, Val5]-Ang II was undetectable in the plasma, it seems that [Asn1, Val5]-Ang II is not a native hormone in lamprey.
To investigate the possible interspecific effect of LpAng II, cannulated Japanese eel was used for comparison. The eel responded to [Asn1, Val5]-Ang II injection with a typical vasopressor response but not to LpAng II. According to the three-dimensional CPH modeling of LpAng II and [Asn1, Val5]-Ang II ( Nielsen et al. 2010), their molecular configurations are highly different due to the extra amino acids in the N-terminus as well as due to the substitution in the aligned region (See Supplementary Figures 1 and 2, see section on supplementary data given at the end of this article). The preliminary modeling showed that [Asn1, Val5]-Ang II, similar to human Ang II, has a linear structure with a hydrophobic pocket formed by the [Tyr4] and [Pro7] ( Tzakos et al. 2003). On the other hand, the predicted structure of LpAng II is helical shape and shared little structural resemblance to [Asn1, Val5]-Ang II three dimensionally. Deletion of the first five amino acids at the N-terminus of LpAng II greatly affected the three-dimensional structure and abolished the vasoactive properties in lamprey (data not shown). Therefore, it is not surprising that LpAng II did not activate the angiotensin receptors in the eel and thus was not detected by the eel vasopressor bioassay in previous isolation studies ( Rankin et al. 2004, Takei et al. 2004).
In conclusion, we discovered and characterized the native angiotensinogen in lamprey, which suggested that the RAS first evolved in the cyclostomes. The circulating form and level of LpAng II were determined by a newly developed RIA in combination with HPLC fractionation. In Japanese lamprey, LpAng II elicited a biphasic vasoactive response, but [Asn1, Val5]-Ang II was undetectable and not vasoactive. The species-specific action of LpAng II in lamprey and eel showed that the RAS has undergone independent paths in vertebrate evolution. Whether LpAng II may signal through the AT1 and AT2 receptors as in other vertebrates is open to question for future research. Questions do remain on the source and function of [Asn1, Val5, Thr9]-Ang I identified by incubation of plasma and kidney extract in previous studies ( Rankin et al. 2004, Takei et al. 2004). Fishes are capable of absorbing intact proteins through the gut directly into the blood stream ( Georgopoulou et al. 1988). Active feeding river lamprey used in the previous studies may have absorbed the host angiotensinogen and renin into their blood stream, and therefore subsequent incubation of plasma with kidney extract may have generated the host-type Ang I. Alternatively, lamprey has a long history of parasitism on fishes ( Gess et al. 2006); horizontal gene transfer or selective gene exploitation may have introduced some teleost genes into the lamprey genome ( Salzet et al. 2000, Yu et al. 2008). We look forward to the completion of the sea lamprey genome project, so that more information can be put together to analyze the possibility of horizontal gene transfer between lamprey and fishes.
Supplementary data
This is linked to the online version of the paper at http://dx.doi.org/10.1530/JOE-10-0422.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
The present study was supported by a Grant-in-Aid for Basic Research (A) (16207004) to Y T and a postdoctoral fellowship to M K S W from Japan Society for the Promotion of Science.
Acknowledgements
We thank Prof. Norman Woo of the Chinese University of Hong Kong for his critical comments on this manuscript and Dr Jillian Healy of the University of Tokyo for editing the English in the paper.
References
Augustinsson KB, Fänge R, Johnels A & Ostlund E 1956 Histological, physiological, and biochemical studies on the heart of two cyclostomes, hagfish (Myxine) and lamprey (Lampetra). Journal of Physiology 131 257– 276.
Brown JA, Cobb CS, Frankling SC & Rankin JC 2005 Activation of the newly discovered cyclostome renin–angiotensin system in the river lamprey, Lampetra fluviatilis. Journal of Experimental Biology 208 223– 232 doi:10.1242/jeb.01362.
Chen J, Shi YH, Li MY, Ding WC & Niu H 2008 Molecular cloning of liver angiotensinogen gene in ayu (Plecoglossus altivelis) and mRNA expression changes upon Aeromonas hydrophila infection. Fish & Shellfish Immunology 24 659– 662 doi:10.1016/j.fsi.2008.01.015.
Cheong HS, Chang JS, Park JM & Byun SY 1990 Affinity enhancement of bispecific antibody against two different epitopes in the same antigen. Biochemical and Biophysical Research Communications 173 795– 800 doi:10.1016/S0006-291X(05)80857-5.
Cobb CS, Williamson R & Brown JA 1999 Angiotensin II-induced calcium signalling in isolated glomeruli from fish kidney (Oncorhynchus mykiss) and effects of losartan. General and Comparative Endocrinology 113 312– 321 doi:10.1006/gcen.1998.7209.
Cobb CS, Frankling SC, Rankin JC & Brown JA 2002 Angiotensin converting enzyme-like activity in tissues from river lamprey or lampern, Lampetra fluviatilis, acclimated to freshwater and seawater. General and Comparative Endocrinology 127 8– 15 doi:10.1016/S0016-6480(02)00014-X.
Donald JA 1998 Autonomic nervous system. In The Physiology of Fishes, 2nd edn, pp 407–439. Ed. DH Evans. Boca Raton, FL: CRC Press..
Ehrlich PH & Moyle WR 1984 Specificity considerations in cooperative immunoassays. Clinical Chemistry 30 1523– 1532.
Ehrlich PH, Moyle WR, Moustafa ZA & Canfield RE 1982 Mixing two monoclonal antibodies yields enhanced affinity for antigen. Journal of Immunology 128 2709– 2713.
Evans DH & Harrie AC 2001 Vasoactivity of the ventral aorta of the American eel (Anguilla rostrata), Atlantic hagfish (Myxine glutinosa), and sea lamprey (Petromyzon marinus). Journal of Experimental Zoology 289 273– 284 doi:10.1002/1097-010X(20010415/30)289:5<273::AID-JEZ1>3.0.CO;2-L.
Falck BV, Mecklenburg C, Myhrberg H & Persson H 1966 Studies on adrenergic and cholinergic receptors in the isolated hearts of Lampetra fluviatilis (Cyclostomata) and Pleuronectes platessa (Teleostei). Acta Physiologica Scandinavica 68 64– 71 doi:10.1111/j.1748-1716.1966.tb03403.x.
Farrel AP 2007 Cardiovascular systems in primitive fishes. In Fish Physiology: Primitive Fishes, vol 26, pp 54–121. Eds DJ McKenzie, AP Farrell & CJ Brauner. San Diego, CA: Academic Press..
Georgopoulou U, Dabrowski K, Sire MF & Vernier JM 1988 Absorption of intact proteins by the intestinal epithelium of trout, Salmo gairdneri. Cell and Tissue Research 251 145– 152 doi:10.1007/BF00215459.
Gess RW, Coates MI & Rubidge BS 2006 A lamprey from the Devonian period of South Africa. Nature 443 981– 984 doi:10.1038/nature05150.
Hopp TP & Woods KR 1981 Prediction of protein antigenic determinants from amino acid sequences. PNAS 78 3824– 3828 doi:10.1073/pnas.78.6.3824.
Irving JA, Cabrita LD, Kaiserman D, Worrall MW & Whisstock JC 2007 Evolution and classification of the Serpin superfamily. In Molecular and Cellular Aspects of the Serpinopathies and Disorders in Serpin Activity, pp 1–33. Eds GA Silverman & DA Lomas. Singapore: World Scientific Publishing..
Khosla MC 1985 Synthesis and pharmacology of non-mammalian angiotensins and their evolutionary development. Peptides 6 ( Suppl 3) 289– 293 doi:10.1016/0196-9781(85)90388-2.
Kobayashi H & Takei Y 1996 The renin–angiotensin system – comparative aspects. In Zoophysiology, vol 35. Berlin: Springer Verlag..
Lignon JM 1979 Responses to sympathetic drugs in the ammocoete heart: probable influence of the small intensely fluorescent (SIF) cells. Journal of Molecular and Cellular Cardiology 11 447– 465 doi:10.1016/0022-2828(79)90469-3.
Marchalonis JJ 1969 An enzymic method for trace iodination of immunoglobulins and other proteins. Biochemical Journal 113 299– 305.
Moyle WR, Lin C, Corson RL & Enrlich RH 1983a Quantitative explanation for increased affinity shown by mixtures of monoclonal antibodies: importance of a circular complex. Molecular Immunology 20 439– 452 doi:10.1016/0161-5890(83)90025-1.
Moyle WR, Anderson DM & Ehrlich PH 1983b A circular antibody–antigen complex is responsible for increased affinity shown by mixtures of monoclonal antibodies to human chorionic gonadotropin. Journal of Immunology 131 1900– 1905.
Nakamura Y, Nishimura H & Khosla MC 1982 Vasodepressor action of angiotensin in conscious chickens. American Journal of Physiology 243 H456– H462.
Nielsen M, Lundegaard C, Lund O & Petersen TN 2010 CPH models-3.0 – remote homology modeling using structure guided sequence profiles. Nucleic Acids Research 38 W576– W581 doi:10.1093/nar/gkq535.
Nishimura H 1985 Evolution of the renin–angiotensin system and its role in control of cardiovascular function in fishes. In Evolutionary Biology of Primitive Fishes, pp 275–293. Eds RE Foreman, A Gorbman, JM Dodd & R Olsson. New York, NY: Plenum Press..
Nishimura H, Oguri M & Ogawa M 1970 Absence of renin in kidneys of elasmobranchs and cyclostomes. American Journal of Physiology 218 911– 915.
Nishimura H, Nakamura Y, Sumner RP & Khosla MC 1982 Vasopressor and depressor actions of angiotensin in the anesthetized fowl. American Journal of Physiology. Heart and Circulatory Physiology 242 H314– H324.
Nishimura H, Walker OE, Patton CM, Madison AB, Chiu AT & Keiser J 1994 Novel angiotensin receptor subtype in fowl. American Journal of Physiology. Regulatory, Integrative and Comparative Physiology 267 R1174– R1181.
Olson KR 1992 Blood and extracellular fluid volume regulation: role of the renin–angiotensin, kallikrenin–kinin systems and atrial natriuretic peptides. In Fish Physiology, vol 12B, pp 135–254. Eds WS Hoar, DJ Randall & AP Farrell. San Diego, CA: Academic Press..
Paul M, Mehr AP & Kreutz R 2006 Physiology of local renin–angiotensin systems. Physiological Reviews 86 747– 803 doi:10.1152/physrev.00036.2005.
Rankin JC, Cobb CS, Frankling SC & Brown JA 2001 Circulating angiotensins in the river lamprey, Lampetra fluviatilis, acclimated to freshwater and seawater: possible involvement in the regulation of drinking. Comparative Biochemistry and Physiology 129B 311– 318 doi:10.1016/S1096-4959(01)00336-0.
Rankin JC, Watanabe TX, Nakajima K, Broadhead C & Takei Y 2004 Identification of angiotensin I in a cyclostome, Lampetra fluiatilis. Zoological Science 21 173– 179 doi:10.2108/zsj.21.173.
Ronquist F & Huelsenbeck JP 2003 MRBAYES3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19 1572– 1574 doi:10.1093/bioinformatics/btg180.
Salzet M, Capron A & Stefano GB 2000 Molecular crosstalk in host–parasite relationships: schistosome- and leech–host interactions. Parasitology Today 16 12 doi:10.1016/S0169-4758(00)01787-7.
Shiels HA, Vornanen M & Farrell AP 2002 Force–frequency relationship in fish heart – a review. Comparative Biochemistry and Physiology 132A 811– 826.
Takei Y, Joss JMP, Kloas W & Rankin JC 2004 Identification of angiotensin I in several vertebrate species: its structural and functional evolution. General and Comparative Endocrinology 135 286– 292 doi:10.1016/j.ygcen.2003.10.011.
Tamura K, Dudley J, Nei M & Kumar S 2007 MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24 1596– 1599 doi:10.1093/molbev/msm092.
Tierney ML, Gramb M & Hazon N 1995a Stimulation of the renin–angiotensin system and drinking by papaverine in the seawater eel, Anguilla anguilla. Journal of Fish Biology 46 721– 724 doi:10.1111/j.1095-8649.1995.tb01107.x.
Tierney ML, Takei Y & Hazon N 1995b A radioimmunoassay for the determination of angiotensin II in elasmobranch fish. General and Comparative Endocrinology 111 299– 305 doi:10.1006/gcen.1998.7114.
Tsuchida T & Takei Y 1998 Effects of homologous atrial natriuretic peptide on drinking and plasma ANG II level in eels. American Journal of Physiology 275 R1605– R1610.
Tzakos AG, Bonvin AMJJ, Troganis A, Cordopatis P, Amzel ML, Gerothanassis IP & van Nuland NAJ 2003 On the molecular basis of the recognition of angiotensin II (AII). NMR structure of AII in solution compared with the X-ray structure of AII bound to the mAb Fab131. European Journal of Biochemistry 270 849– 860 doi:10.1046/j.1432-1033.2003.03441.x.
Vornanen M, Shiels HE & Farrell AP 2002 Plasticity of excitation–contraction coupling in fish cardiac myocytes. Comparative Biochemistry and Physiology 132A 827– 846 doi:10.1016/S1095-6433(02)00051-X.
Watanabe T, Inoue K & Takei Y 2009 Identification of angiotensinogen genes with unique and variable angiotensin sequences in chondrichthyans. General and Comparative Endocrinology 161 115– 122 doi:10.1016/j.ygcen.2008.11.021.
Wong MKS & Takei Y 2009 Cyclostome and chondrichthyan adrenomedullins reveal ancestral features of the adrenomedullin family. Comparative Biochemistry and Physiology 154B 317– 325 doi:10.1016/j.cbpb.2009.07.006.
Wong MKS, Ge W & Woo NYS 2007 Positive feedback of hepatic angiotensinogen expression in silver sea bream (Sparus sarba). Molecular and Cellular Endocrinology 263 103– 111 doi:10.1016/j.mce.2006.09.001.
Yu F, Li Y, Liu L & Li Y 2008 Comparative genomics of human-like Schistosoma japonicum genes indicate a putative mechanism for host–parasite relationship. Genomics 91 152– 157 doi:10.1016/j.ygeno.2007.10.006.
