Differential protein expression of mitogen-activated protein kinases in erythrocytes and liver of lamprey Lampetra fluviatilis on the course of prespawning starvation.

The study was designed to identify the types of mitogen-activated protein kinases (MAPKs) in erythrocytes and liver tissues of river lamprey Lampetra fluviatilis and monitor the changes in protein expression levels of found enzymes on the course of prespawning starvation (from November to the end of May). Immunoreactivity of the native and phosphorylated forms of ERK1/2, JNK and p38 was examined in the cytosolic and membrane cell fractions. Both lamprey erythrocytes and liver were found to highly express ERK1/2 and JNK, whereas only trace amounts of p38 were revealed in hepatic tissues. ERK1/2 was identified in cytosolic and membrane fractions, whereas JNK and p38 were predominantly cytosolic enzymes. Total cellular amounts of ERK1/2 and phospho-ERK1/2 in both erythrocytes and liver tissues appeared to be relatively stable on the course of prespawning starvation. However, before spawning ERK1/2 translocated from cytosol to membranes, with partial decline of its cytoplasmic expression being compensated by increases in membrane-bound pool. Immunoreactivity of cytoplasmic JNK, phospho-JNK and p38 were stable from November to March, but sharply decreased before spawning exhibiting almost negligible levels in May, which suggests the depletion of their cellular fractions. Most probably, ERK1/2 plays more important role in mediating adaptive responses of erythrocytes and liver tissues to conditions of natural starvation and maintenance of cell viability before spawning and death of animals in May.

Molecular Evolution of Apolipoprotein Multigene Family and the Original Functional Properties of Serum Apolipoprotein (LAL2) in Lampetra japonica.

Apolipoprotein (APO) genes represent a large family of genes encoding various binding proteins associated with plasma lipid transport. Due to the long divergence history, it remains to be confirmed whether these genes evolved from a common ancestor through gene duplication and original function, and how this evolution occurred. In this study, based on the phylogenetic tree, sequence alignment, motifs, and evolutionary analysis of gene synteny and collinearity, APOA, APOC, and APOE in higher vertebrates may have a common ancestor, lamprey serum apolipoprotein LAL1 or LAL2, which traces back to 360 million years ago. Moreover, the results of immunofluorescence, immunohistochemistry, and flow cytometry show that LAL2 is primarily distributed in the liver, kidney, and blood leukocytes of lampreys, and specifically localized in the cytoplasm of liver cells and leukocytes, as well as secreted into sera. Surface plasmon resonance technology demonstrates that LAL2 colocalizes to breast adenocarcinoma cells (MCF-7) or chronic myeloid leukemia cells (K562) associated with lamprey immune protein (LIP) and further enhances the killing effect of LIP on tumor cells. In addition, using quantitative real-time PCR (Q-PCR) and western blot methods, we found that the relative mRNA and protein expression of lal2 in lamprey leukocytes and sera increased significantly at different times after stimulating with Staphylococcus aureus, Vibrio anguillarum, and Polyinosinic-polycytidylic acid (Poly I:C). Moreover, LAL2 was found to recognize and bind to gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) and gram-negative bacteria (Escherichia coli) and play an important role in the antibacterial process. All in all, our data reveals a long, complex evolutionary history for apolipoprotein genes under different selection pressures, confirms the immune effect of LAL2 in lamprey sera against pathogens, and lays the foundation for further research regarding biological functions of lamprey immune systems.

Evolutionary conservation of the allosteric activation of factor VIIa by tissue factor in lamprey.

Essentials Tissue factor (TF) enhances factor VIIa (FVIIa) activity through structural and dynamic changes. We analyzed conservation of TF-activated FVIIa allosteric networks in extant vertebrate lamprey. Lamprey Tf/FVIIa molecular dynamics show conserved Tf-induced structural/dynamic FVIIa changes. Lamprey Tf activation of FVIIa allosteric networks follows molecular pathways similar to human. SUMMARY: Background Previous studies have provided insight into the molecular basis of human tissue factor (TF) activation of activated factor VII (FVIIa). TF-induced allosteric networks of FVIIa activation have been rationalized through analysis of the dynamic changes and residue connectivities in the human soluble TF (sTF)/FVIIa complex structure during molecular dynamics (MD) simulation. Evolutionary conservation of the molecular mechanisms for TF-induced allosteric FVIIa activation between humans and extant vertebrate jawless fish (lampreys), where blood coagulation emerged more than 500 million years ago, is unknown and of considerable interest. Objective To model the sTf/FVIIa complex from cloned Petromyzon marinus lamprey sequences, and with comparisons to human sTF/FVlla investigate conservation of allosteric mechanisms of FVIIa activity enhancement by soluble TF using MD simulations. Methods Full-length cDNAs of lamprey tf and f7 were cloned and characterized. Comparative models of lamprey sTf/FVIIa complex and free FVIIa were determined based on constructed human sTF/FVIIa complex and free FVIIa models, used in full-atomic MD simulations, and characterized using dynamic network analysis approaches. Results Allosteric paths of correlated motion from Tf contact points in lamprey sTf/FVIIa to the FVIIa active site were determined and quantified, and were found to encompass residue-residue interactions along significantly similar paths compared with human. Conclusions Despite low conservation of residues between lamprey and human proteins, 30% TF and 39% FVII, the structural and protein dynamic effects of TF activation of FVIIa appear conserved and, moreover, present in extant vertebrate proteins from 500 million years ago when TF/FVIIa-initiated extrinsic pathway blood coagulation emerged.

[PHOSPHOLIPIDS AND FATTY ACIDS IN ERYTHROCYTES OF THE LAMPREY LAMPETRA FLUVIATILIS DURING AUTUMN PRESPAWNING PERIOD AND THE ABSORPTION SPECTRUM OF THEIR LIPID EXTRACT].

The content of some classes of phospholipids and their fatty acid composition in erythrocytes of the lamprey Lampetrafluviatilis during the autumn period of its prespawning migration are investigated. It is found that the phospholipid spectrum of erythrocytes of the lamprey, the oldest representative of vertebrates, is similar to that of many mammals. A four-fold prevalence of phosphatidilcholine content over sphingomyelin content as well as prevalence of (ω3-acids over ω6-acids indicates the <> of lamprey's erythrocyte membranes - an important indicator of deformational ability of lamprey's erythrocytes. Phosphatidilethanol amine and its plasmalogenic form are the most unsaturated phospholipids (their unsaturation indices are 230 and 342, correspondingly). Phosphatidilcholine is the most saturated one (UI is 167). It is found that the basic acid indicators characterizing the fluidity of erythrocyte membranes remain unchanged during the whole period of prespawning migration of lampreys up to spawning. The blood contains several buffer systems, in particular, membrane phospholipids which neutralize acids and alkali incoming into the blood. In the process of organism life a change of pH inside erythrocytes occurs. One can suppose that the base of the system associated with buffer properties of the blood is water dissociation. Inside thin vessels of the circulatory system the hemoglobin attaches and returns molecules of oxygen due to interaction of the buffer systems with water. The property of water to dissociate as well as ion transfer produce in erythrocytes, lying within narrow vessels of the circulatory system, a local pH alteration allowing displacing/attaching the molecule of oxygen from hemoglobin.

Bioinformatic Characterization of Genes and Proteins Involved in Blood Clotting in Lampreys.

Lampreys and hagfish are the earliest diverging of extant vertebrates and are obvious targets for investigating the origins of complex biochemical systems found in mammals. Currently, the simplest approach for such inquiries is to search for the presence of relevant genes in whole genome sequence (WGS) assemblies. Unhappily, in the past a high-quality complete genome sequence has not been available for either lampreys or hagfish, precluding the possibility of proving gene absence. Recently, improved but still incomplete genome assemblies for two species of lamprey have been posted, and, taken together with an extensive collection of short sequences in the NCBI trace archive, they have made it possible to make reliable counts for specific gene families. Particularly, a multi-source tactic has been used to study the lamprey blood clotting system with regard to the presence and absence of genes known to occur in higher vertebrates. As was suggested in earlier studies, lampreys lack genes for coagulation factors VIII and IX, both of which are critical for the "intrinsic" clotting system and responsible for hemophilia in humans. On the other hand, they have three each of genes for factors VII and X, participants in the "extrinsic" clotting system. The strategy of using raw trace sequence "reads" together with partial WGS assemblies for lampreys can be used in studies on the early evolution of other biochemical systems in vertebrates.

Simultaneous determination of gonadotropin-inhibitory and gonadotropin-releasing hormones using ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry.

Gonadotropin-inhibitory hormones (GnIH) and gonadotropin-releasing hormones (GnRH) are neuropeptides essential for the regulation of reproduction in all vertebrate animals examined. Determination of neuropeptides in the biological sample is highly challenging due to their complex matrix and weak stability. The wide variety of peptides or protein degradation products often interferes with the determination of the target peptide. This study aims to develop a specific ultra-high performance liquid chromatography-tandem mass spectrometry method for simultaneous determination of nine critical neuropeptides in biological samples. A separation method by ultra-performance liquid chromatography coupled to a multiple reaction monitoring (MRM) by tandem mass spectrometry allows the selective determination of the neuropeptides in brain and plasma matrices after solid-phase extraction. Specific MSMS transitions were optimized using MRM of multiple-charged peptides generated by electrospray ionization in positive mode. The resulting analytical method was fully validated with thorough evaluation of stability, recovery, matrix effect, and intra- and interday accuracy and precision in sea lamprey brain and plasma. The optimized method shows linearity in a wide range of concentrations with limit of quantification ranging from 0.1 to 0.75 ng/mL. With slight modification, this method can be applied to other biological samples.

[Liver monoamine oxidase activity of the lamprey Lampetra fluviatilis. the substrate-inhibitory specificity].

Based on data of substrate-inhibitory analysis with use of specific inhibitors--deprenyl, chlorgi-lin--and specific substrates--serotonin, noradrenalin, benzylamine, beta-phenylethylamine, and N-methylhistamine--a suggestion is put forward about the possible existence of one molecular form of monoamine oxidase (MAO) in liver of mature individuals of the European lamprey Lampetra fluviatilis. There are determined kinetic parameters of monoamine oxidase deamination of eight substrates, which indicates the large spectrum of substrate specificity of the lamprey liver MAO. The studied enzyme does not deaminate histamine and putrescine and is not sensitive to 10(-2) M semicarbaside. Results of study of the substrate-inhibitor specificity allow us to suggest some resemblance of catalytic properties of the lamprey liver MAO and the mammalian form A MAO. The revealed low activity of the enzyme at deamination of all used substrates seems to be connected with low detoxational functional of the lamprey liver.

Characterization, phylogenetic analysis and cDNA cloning of natterin-like gene from the blood of lamprey, Lampetra japonica.

Lamprey as a "living fossil" of immunological origin and "rich treasure" of biological pharmaceutical development has caused attention of scholars. The cDNA library construction and EST sequencing of blood had been done previously in our lab, and bioinformatics analysis provided a gene fragment which is highly homologous with natterin family, named natterin-like. To elucidate the characterization and phylogeny of natterin-like genes in early evolution, we cloned the full-length cDNA of natterin-like gene from the blood of Lampetra japonica. The open reading frame of this sequence contained 942bp and encoded 313 amino acids, including a lectin-like domain and a pore-forming toxin-like domain. Using reverse transcription PCR, natterin-like mRNA was also detected in lamprey blood, kidney, heart, liver, medullary, gonad, but absent in lamprey intestine and gill. Our results suggested that in lampreys and most of other species, there might be only one natterin-like gene, which was fused by certain sequences during evolution and encoded proteins with more functions. It is similar between C terminal of natterin-like protein and Aerolysin in space structure and the lectin-like domain of natterin-like equivalent to glycoprotein binding motif of Aerolysin in function. We also propose that the defense mechanism against specific predators in historical evolution of lamprey. Our findings may provide insights into the function and characterization of natterin-like genes as well as other gene families in vertebrates and provide a foundation for identification and structural, functional, and evolutionary analyses of more natterin-like genes and other gene families.

The presence of teleost-type angiotensin components in lamprey buccal gland suggests a role in endocrine mimicry.

Previous characterization of a native lamprey angiotensin II (LpAng II) that possesses a different sequence and function than teleost-type angiotensin II (Ang II) has raised a question as to the role of teleost-type angiotensin peptides in lampreys. In this study, teleost-type angiotensin like-peptides were identified in the buccal gland of lampreys by immunoassays and immunohistochemistry. The possible sources of angiotensin like-peptides were investigated in lampreys by manipulating their choice of host and food. Ang II immunoreactivity (irAng II) was detected in the buccal gland and plasma of feeding phase sea lampreys exposed to Atlantic cod, but was mostly absent in fasting lamprey. Qualitatively, the HPLC profiles of irAng II observed in the plasma, when present, were highly similar to those in buccal gland, implying that the buccal gland could be a source of plasma Ang II. Japanese lampreys force-fed with dogfish blood had significantly elevated concentrations of irAng II in their buccal glands when compared to unfed individuals, suggesting that feeding stimuli may have enhanced buccal gland activity. Teleost-type Ang II-containing proteins, other than angiotensinogen, are present in the buccal gland as trypsinization generated Ang II in vitro, and the HPLC profile of these irAng II was highly comparable to those naturally present in the buccal gland. [Asn(1), Val(5), Thr(9)]-Ang I that was identified in the buccal gland of Japanese lampreys has the same amino acid sequence to those previously isolated from the incubation of plasma and kidney extract, providing an alternative explanation for the previous isolation of teleost-type Ang I in lampreys. irAng I and irAng II were localized in the granule-like structures in the apical region of the secretory epithelia, suggesting that these peptides may be active components of lamphredin. The teleost-type angiotensin peptides in the buccal gland secretion suggested that these host-specific peptides could be part of the endocrine mimicry strategy used by lampreys to evade host immune responses and reduce immune-rejection.

Evolutionary origin of the vertebrate blood complement and coagulation systems inferred from liver EST analysis of lamprey.

The complement and coagulation systems in mammalian blood are composed of multiple components with unique domain structures, and are believed to be established by exon-shufflings and following gene duplications. To elucidate their origin in vertebrates, liver EST and 5'- and 3'-rapid amplification of cDNA ends (RACE) analyses were performed in lamprey, Lethenteron japonicum. For the complement system, thefactor I cDNA was cloned for the first time outside of the jawed vertebrates. Evidence for the C3/C4/C5, fB/C2 and MASP-1/MASP-2/C1r/C1s gene duplications was not found, suggesting that these duplications occurred in the jawed vertebrate lineage. In contrast, the coagulation factors VII and X, prothrombin and protein C-like cDNAs were identified, indicating that duplications among them predated the cyclostome-jawed vertebrate divergence. The genes for terminal complement components, coagulation factors XI and XII, or prekallikrein were not found, suggesting that the complement and coagulation systems of an ancestral vertebrate were simpler compared to their mammalian counterparts.

Glucose and free amino acids in the blood of lampreys (Lampetra fluviatilis L.) and frogs (Rana temporaria L.) under prolonged starvation.

The content variation dynamics of glucose and free amino acids in blood plasma was followed for lampreys and frogs from autumn till spring, when the exogenous feeding is switched off. In October, the glucose level is rather high (8-10 mM) in blood plasma of both lampreys and frogs. It falls by 50% during winter and falls to a critically low level (1-2 mM) during spring. The lamprey plasma amino acid levels increase by 74% from November to April and thus reach the lower limit known for mammals. The amount of free amino acids in frog plasma decreases by 40% by spring in comparison with the values in autumn. More intensive proteolysis in lamprey tissues compared with that in frog tissues has been confirmed by quantitatively determining leucine, isoleucine, and valine in the blood of these animals. Besides these three amino acids, alanine, glycine, lysine, threonine and, in certain periods, tyrosine have been found to be quantitatively significant in the plasma of both animals.

Amiloride-sensitive sodium transport in lamprey red blood cells: evidence for two distinct transport pathways.

To determine Na+/H+ exchange in lamprey erythrocyte membranes, the cells were acidified to pH(i) 6.0 using the K+/H+ ionophore nigericin. Incubation of acidified erythrocytes in a NaCl medium at pH 8.0 caused a considerable rise in 22Na+ influx and H+ efflux during the first 1 min of exposure. In addition, exposure of acidified red cells to NaCl medium was associated with rapid elevation of intracellular Na+ content. The acid-induced changes in Na+ influx and H+ efflux were almost completely inhibited by amiloride and dimethylamiloride. In native lamprey erythrocytes, amiloride-sensitive Na+ influx progressively increased as the osmolality of incubation medium was increased by addition of 100, 200, or 300 mmol/l sucrose. Unexpectedly, the hypertonic stress induced a small, yet statistically significant decrease in intracellular Na+ content in these cells. The reduction in the cellular Na+ content increased with hypertonicity of the medium. The acid- and shrinkage-induced Na+ influxes were inhibited by both amiloride and 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) in a dose-dependent manner. For both blockers, the half-maximal inhibitory values (IC50) were much greater for the shrinkage-induced (44 and 15 micromol/l for amiloride and EIPA, respectively) than for the acid-induced Na+ influx (5.1 and 3.3 micromol/l, respectively). The data obtained are the first demonstration of the presence of a Na+/H+ exchanger with high activity in acidified (pH(i) 6.0) lamprey red blood cells (on average, 512 +/- 56 mmol/l cells/h, n = 13). The amiloride-sensitive Na+ influxes produced by hypertonic cell shrinkage and acid load are likely to be mediated by distinct ion transporters in these cells.

Effect of alloxan and insulin immunoneutralization on circulating thyroid hormone levels in larval landlocked sea lampreys, petromyzon marinus.

The effects of alloxan, an insulin (INS)-secreting cell toxin, and INS immunoneutralization on circulating levels of thyroid hormones (thyroxine, T(4); triiodothyronine, T(3)) were examined in larval landlocked sea lampreys, Petromyzon marinus. Animals were injected intraperitoneally with either (Experiment 1) saline (0.6%) or alloxan (20 or 200 microg/g body weight), or with (Experiment 2) normal rabbit serum or anti-lamprey INS. Alloxan (200 microg/g) decreased plasma T(3), but not T(4), in larvae by about 45-80%. Three, six, or nine hr after acute immunoneutralization of lamprey INS with anti-lamprey INS, plasma T(3) levels decreased by 13-30%, relative to controls. These data indicate that INS deficiency can regulate the thyroid system of larval lampreys. There is some suggestion that INS may mediate the metamorphic processes by modulating thyroid hormone concentrations.

Blood steroid profile and in vitro steroidogenesis by ovarian follicles and testis fragments of adult sea lamprey, Petromyzon marinus.

The main purpose of the study was to identify the principal gonadal steroids synthesized by male and female sea lampreys, Petromyzon marinus. To achieve this, we used high performance liquid chromatography to separate the steroids in the serum of sexually mature animals, and to separate the steroids produced by gonadal tissue incubated in the presence of radiolabelled precursor steroids, as a means of identifying the major steroidogenic pathways. We were unable to detect evidence of the 'classical' steroids, such as 17beta-estradiol (E(2)) or testosterone (T) in the serum of either male or female lampreys. Instead, the principal chromatographic peaks contained very polar compounds that had elution times consistent with 15alpha-hydroxylated estrogens and androgens, and there were sex-specific differences in the chemical nature and the quantity of these compounds. Testis fragments or ovarian follicles co-incubated with tritium-labelled pregnenolone ([3H]P(5)), 17-hydroxyprogesterone ([3H]17OHP(4)), or androstenedione ([3H]A(4)), provided additional confirmation that the gonads synthesize a range of very polar steroids, and the metabolites found were consistent with the presence of a 15alpha-hydroxylated (15alphaOH) metabolic pathway common to testis and ovary. For ovarian tissue, the major 'end product' metabolites from all three precursors were 15alphaOH-estrogens, and for testis tissue 15alpha-hydroxyprogesterone (15alphaOHP(4)) and 15alpha-hydroxytestosterone (15alphaOHT) and small amounts of 15alphaOH estrogen. Small amounts of E(2) were also produced by both ovarian (all substrates) and testicular tissue (some substrates). Although it was assumed that the E(2) was synthesized via the aromatization of T, [3H]T was not found as an intermediate metabolite. The study suggests that the principal gonadal steroids in sea lamprey are 15alpha-OH compounds, and that only small amounts of E(2) or T are synthesized by the gonads at this stage of reproductive development. There was no direct evidence of progesterone (P(4)) synthesis from [3H]P(5), although the metabolites synthesized by both testis and ovary were indicative of a metabolic pathway that involved P(4) as an intermediate.

ATP-sensitive K+ and Ca2+-activated K+ channels in lamprey ( Petromyzon marinus) red blood cell membrane.

The patch-clamp technique was used to demonstrate the presence of ATP-sensitive K(+) channels and Ca(2+)-activated K(+) channels in lamprey ( Petromyzon marinus) red blood cell membrane. Whole-cell experiments indicated that the membrane current under isosmotic (285 mosmol l(-1)) conditions is carried by K(+). In the inside-out configuration an ATP-sensitive K(+) channel (70-80 pS inward, 35-40 pS outward) was present in 35% of patches. Application of ATP to the intracellular side reduced unitary current with half-maximal inhibition in the range 10-100 microM. A block was obtained with 100 microM lidocaine and inhibition was obtained with 0.5 mM barium acetate. A Ca(2+)-activated K(+) channel (25-30 pS inward, 10-15 pS outward) was present in 57% of patches. Inhibition was produced by 10 mM TEA and 500 nM apamin and sensitivity to Ba(2+) was lower than for ATP-sensitive channels. No spontaneous channel activity was recorded in the cell-attached configuration under isotonic conditions. With hypotonic saline 68% of patches showed spontaneous single-channel activity, and, of 75 active patches, 66 cell-attached patches showed channel activity corresponding to Ca(2+)-activated K(+) channels.

Comparative analysis of autoxidation of haemoglobin.

Autoxidation of oxyhaemoglobin (oxyHb) to methaemoglobin was measured at different temperatures in haemoglobin solutions from Atlantic hagfish, river lamprey, common carp, yellowfin tuna and pig. The aims were to evaluate the impact of the absent distal histidine in hagfish haemoglobin, the importance of oxyHb being either monomeric (hagfish and lamprey) or tetrameric (carp, tuna and pig) and to gain information on the temperature-sensitivity of autoxidation. The rate of autoxidation was lower in hagfish than in carp, yellowfin tuna and lamprey haemoglobins at any given temperature. Substitution of the distal histidine residue (His E7) with glutamine in hagfish haemoglobin was therefore not associated with an accelerated autoxidation, as might be expected on the basis of the normal protective role of His E7. Glutamine may have similar qualities to histidine and be involved in the low susceptibility to autoxidation. The low oxidation rate of hagfish haemoglobin, together with an oxidation rate of lamprey haemoglobin that did not differ from that of carp and yellowfin tuna haemoglobins, also revealed that autoxidation was not accelerated in the monomeric oxyhaemoglobins. Pig haemoglobin was oxidised more slowly than fish haemoglobins, demonstrating that fish haemoglobins are more sensitive to autoxidation than mammalian haemoglobins. The rate of autoxidation of hagfish haemoglobin was, however, only significantly greater than that of pig haemoglobin at high temperatures. Autoxidation was accelerated by rising temperature in all haemoglobins. Arrhenius plots of carp and yellowfin tuna haemoglobin revealed a break at 25 degrees C, reflecting a lower temperature-sensitivity between 5 and 25 degrees C than between 25 and 40 degrees C.

Circulating angiotensins in the river lamprey, Lampetra fluviatilis, acclimated to freshwater and seawater: possible involvement in the regulation of drinking.

Plasma angiotensin levels were measured for the first time in a cyclostome, the river lamprey. With the demonstration that angiotensins are present in the circulation, the possibility of a physiological role in the regulation of drinking was re-examined. Angiotensin II and III concentrations and plasma osmolalities were significantly higher in lampreys acclimated to 28 ppt seawater than in those acclimated to freshwater. No changes were found in angiotensin II and III levels 4 h after transfer from freshwater to 50% seawater, although plasma osmolality had started to rise by this time. There was a suggestion that plasma angiotensin II levels might be related to osmolality in the transfer experiment. Injection of Asp(1)Val(5)- or Asn(1)Val(5)-angiotensin II (40-169 microg/kg body wt.) did not stimulate drinking in freshwater-acclimated lampreys, even when they were still capable of drinking. The angiotensin-converting enzyme inhibitor captopril and the smooth muscle relaxant papaverine both reduced drinking rate in 50% seawater-acclimated lampreys. The data do not provide direct evidence for the involvement of the renin-angiotensin system in the control of drinking behaviour in the lamprey. Indirect evidence from the captopril effect is suggestive, but could have other explanations.

Unique distribution of the anion exchange protein in the sea lamprey, Petromyzon marinus.

The purpose of the present study was to examine the distribution of the anion exchange protein in the sea lamprey. Southern blots showed that genomic DNA of juvenile lampreys possesses several regions that are similar to segments of the AE gene from other vertebrates. However, physiological experiments examining rapid anion fluxes across the red blood cell (rbc) membrane and molecular experiments examining mRNA transcript levels both indicated that the anion exchange protein is absent in sea lamprey rbcs. In contrast, lamprey kidney, skeletal muscle, liver and heart tissue all appeared to possess mRNA transcripts for an AE protein. Further evidence for the presence of an AE protein in kidney tissue was obtained from Western blots. In order to evaluate the impact of the apparent rbc anion exchange limitations, the bicarbonate permeability of lamprey rbcs was also evaluated using mass spectrometry. The bicarbonate permeability of the lamprey rbc membrane was an order of magnitude lower than that of trout rbcs. Taken together, these results indicate that the gene for the AE protein is indeed present in lampreys, but it is not expressed in the rbc. Moreover, the process of CO2 transport in lamprey probably does not involve bicarbonate transport across the rbc membrane.