INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

BRADYKININ (BK) is an active component of the kallikrein-kinin system that exhibits profound actions on smooth muscle contraction and vascular permeability in mammals (22). In nontetrapod species, several molecular forms of BK have been isolated from the plasma of actinopterygian (sturgeon, gar, bowfin, cod, and trout) as well as sarcopterygian (lungfish) bony fish, but their functions have not been fully elucidated (reviewed in Ref. 3). The kallikrein-kinin system has attracted the attention of basic and clinical researchers in relation to the renin-angiotensin system, because both systems utilize a common enzyme, ANG I-converting enzyme (ACE) or kininase II, which is responsible for production of ANG II and degradation of BK (9).

Various types of ACE inhibitors have been used to prevent activation of the renin-angiotensin system. However, their application often produces unexpected results that cannot be explained solely by the inhibition of ANG II formation. In a previous study, we found that captopril infused at low doses resulted in marked depression of drinking and arterial pressure in seawater-adapted eels (30). Because immunoneutralization of plasma ANG II with a specific antiserum did not affect drinking and arterial pressure in the eels, the effects of captopril may not be due to the inhibition of ANG II formation in plasma. In mammals, BK is known to be dipsogenic and vasodepressor when given systemically in combination with captopril (8). If this is also the case in the eel, the vasodepressor effect of captopril observed in the previous study may be mediated by an increase in plasma BK concentrations. However, the cardiovascular effects of BK in teleost fishes are species dependent (19, 20), and there appears to be no report on the effects of BK on drinking in fish.

The present study was undertaken to examine the effect of BK on drinking and arterial blood pressure in eels. To use homologous BK in the study, we first isolated BK from heat-denatured eel plasma after incubation with porcine pancreatic kallikrein. The amino acid sequence of European eels, Anguilla anguilla, has been determined except for a single residue (7). To assess the type of BK receptors mediating each activity, the effects of selected eel BK analogs (BK for B₂ receptor and [Arg⁰]-des-Arg⁹-BK for B₁ receptor) were also examined. Because bolus injections of eel BK markedly increased arterial pressure, which in itself may affect drinking in eels (12), eel BK was infused slowly at nonpressor doses to mimic more closely the physiological changes expected from activation of the endogenous kallikrein-kinin system. Concentrations of ANG II in plasma were monitored during BK infusion, because ANG II is a potent dipsogen in eels (33).

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animals

Eel BK Sequence

Production of eel BK. Freshwater eels were anesthetized by immersion for 5 min in 0.2% (wt/vol) tricaine methanesulfonate (Sigma, St. Louis, MO) neutralized with sodium bicarbonate. Blood was collected from the caudal vein into a chilled syringe containing 3.8% sodium acetate (10 µl/ml blood). The blood was centrifuged, and plasma was immediately frozen on dry ice. A total of 75 ml of plasma was collected from 30 eels weighing 203.3 ± 2.4 (SE) g. Plasma was thawed and heated to >90°C in 450 ml of 0.2% acetic acid for 30 min to inhibit proteolytic enzyme activity. After the plasma was cooled, 50 ml of 0.5 M Tris · HCl (pH 7.8) containing 1 M NaCl were added to the mixture, and the pH was adjusted to 7.8 with 10 M NaOH. The mixture was incubated with porcine pancreatic kallikrein (1,000 U, Sigma) at 37°C for 90 min with gentle agitation. The reaction was terminated by addition of 1 ml of trifluoroacetic acid (TFA), and the mixture was centrifuged at 40,000 g for 30 min at 4°C to remove the precipitate. The supernatant was passed through activated Sep-Pak C₁₈ cartridges (Waters, Milford, MA), and the adsorbed materials were eluted with 70% acetonitrile-water containing 0.1% (vol/vol) TFA. The eluant was lyophilized and transported to Creighton University for peptide purification studies.

Purification of eel BK. The kallikrein-treated plasma extract, after partial purification on Sep-Pak cartridges, was redissolved in 3 ml of 1% TFA and chromatographed on a 90 × 1.6-cm column of Sephadex G-25 (Pharmacia, Uppsala, Sweden) equilibrated with 1 M acetic acid. The column was eluted at a flow rate of 24 ml/h, and fractions (2 ml) were collected. Absorbance was measured at 280 nm. The concentration of immunoreactive BK in the fractions was determined by radioimmunoassay (15). Fractions containing immunoreactive BK were pooled and injected onto a 25 × 1-cm Vydac 218TP510 (C₁₈) column (Separations Group, Hesperia, CA) equilibrated with 0.1% TFA at a flow rate of 2 ml/min. The concentration of acetonitrile in the eluting solvent was raised to 14% over 10 min and to 35% over 50 min using linear gradients. Absorbance was monitored at 214 and 280 nm, and fractions (2 ml) were collected. The fraction containing immunoreactive BK (Fig. 1A) was sequentially rechromatographed on 25 × 0.46-cm columns of Vydac 214TP54 (C₄), Vydac 219TP54 (phenyl), and Supelcosil LC-18-DB (C₁₈; Supelco Bellefonte, PA) at a flow rate of 1.5 ml/min.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Purification by reverse-phase HPLC of eel [Arg0]bradykinin (BK) on semipreparative Vydac C18 (A), analytic Vydac C4 (B), analytic Vydac phenyl (C), and analytic Supelcosil C18 columns (D). BK denotes the peaks containing BK-like immunoreactivity. Arrows, collection period; dashed line, concentration of acetonitrile in the eluting solvent. ABS214, absorption at 214 nm.

Structural analysis. The primary structure of the peptide was determined by automated Edman degradation using a sequenator (model 471A, Applied Biosystems). Electrospray mass spectrometry was carried out using a single-quadrupole instrument (Sciex API 150EX, Perkin Elmer). The accuracy of mass determinations was ±0.02%.

Peptide synthesis. Eel [Arg⁰]BK (Arg-Arg-Pro-Pro-Gly-Trp-Ser-Pro-Leu-Arg) and its analogs (BK and [Arg⁰]-des-Arg⁹-BK) were synthesized as previously described (13), and their identities was confirmed by amino acid sequencing and mass spectrometry. All peptides were >95% pure.

Physiological Studies

Surgical procedures. A total of 20 seawater-adapted eels were used: 8 for experiment 1 (178.8 ± 4.8 g), 6 for experiment 2 (184.3 ± 8.2 g), and 6 for experiment 3 (183.9 ± 3.1 g). Eels were anesthetized in 0.1% tricaine methanesulfonate for 10 min. In eels used for experiments 1 and 2, vinyl tubes (1.5 mm OD) were inserted into the esophagus and stomach for measurement of drinking rates as described previously (31). In all eels, a polyethylene cannula (0.8 mm OD) was inserted into the dorsal aorta for administration of BK and measurement of blood pressure. In eels used for experiments 2 and 3, another similar cannula was inserted into the ventral aorta for blood sampling. Eels that bled >0.5 ml (7% of total blood volume) were excluded from the study, since these fish usually drink at a higher rate because of hypovolemia and increased plasma ANG II (28). After surgery, the esophageal catheter was connected to a drop counter for continuous measurement of drinking rate, and the stomach catheter was connected to a pulse injector synchronized with the drop counter for reintroduction of the water that had been drunk (31). Eighty percent seawater was reintroduced into the stomach, since ingested seawater that appeared from the esophageal catheter was diluted to this concentration because of a facilitated absorption of Na⁺ and Cl by the esophagus of seawater eels (32). The cannula in the dorsal aorta was connected via a three-way stopcock to a pressure transducer (model DX-300, Nihon Kohden, Tokyo, Japan) for continuous measurement of arterial pressure and to a syringe for injection or infusion in experiments 1 and 2. Eels were allowed to recover for >18 h postoperatively.

Experimental protocol. In experiment 1, eels received bolus injections of eel [Arg⁰]BK, BK, and [Arg⁰]-des-Arg⁹-BK at doses of 1, 3, and 10 nmol/kg in 0.05 ml of vehicle (0.9% NaCl containing 0.01% Triton X-100) over 10 s. Injections of vehicle alone served as controls. Because strong tachyphylaxis occurred for the cardiovascular effects that lasted for several hours after injection of high doses of BK, injections were made from low to high doses at sufficient intervals (1 h). Bolus injections caused acute increases in arterial blood pressure, which probably affected drinking rates (12). In experiments 2 and 3, therefore, [Arg⁰]BK, which was shown to be the most potent peptide in experiment 1, was infused slowly at a rate of 0.6 ml/h (experiment 2) or 0.4 ml/h (experiment 3) to minimize changes in pressure. Infusions were started with vehicle only for 30 min, then increasing doses of [Arg⁰]BK (1, 10, 100, and 1,000 pmol · kg¹ · min¹) and, finally, vehicle were infused for 2 h. To nullify the increase in blood volume caused by the infusion, blood (0.3 or 0.2 ml) was withdrawn every 30 min into a chilled syringe containing 10% (wt/vol) K₂EDTA (10 µl/ml blood). In experiment 2, ANG II concentrations in plasma were measured by radioimmunoassay as described previously (30). The antiserum used for ANG II radioimmunoassay was originally raised against [Asp¹,Ile⁵]ANG II (mammalian ANG II). However, it had ~100% cross-reactivity with [Asn¹,Val⁵]ANG II (eel ANG II) but negligible cross-reactivity with eel angiotensin I (35). The intra- and interassay coefficients of variation were 5.2% and 12.6%, respectively. In experiment 3, an aliquot of blood was transferred to a capillary tube for determination of hematocrit. The remaining blood was centrifuged, and plasma was used for determination of plasma Na⁺ concentration by an atomic absorption spectrophotometer (model Z5300, Hitachi, Tokyo, Japan) and plasma osmolality by a vapor pressure osmometer (VAPRO 5520, Wescor, Logan, UT). All determinations were made in duplicate or triplicate.

Analysis of data. The time-course data were analyzed statistically by ANOVA followed by Bonferroni/Dunn's test at each time point. The dose-response relationship was examined by ANOVA followed by a regression analysis. The difference of responses between doses was examined by randomization test for matched pairs (25). The relative potency of three BK peptides ([Arg⁰]BK, BK, and [Arg⁰]-des-Arg⁹-BK) for the antidipsogenic and cardiovascular effect was examined by two-way ANOVA with repeated measures. Significance was determined at P < 0.05. Values are means ± SE.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Eel BK Sequence

Purification of eel BK. The BK-like immunoreactivity in the kallikrein-treated eel plasma was eluted from a Sephadex G-25 column as a single broad peak with maximum immunoreactivity at the elution volume of mammalian BK. The immunoreactive fractions were pooled and injected onto a semipreparative Vydac C₁₈ column (Fig. 1A). Immunoreactive BK was eluted in a single fraction (horizontal bar in Fig. 1A). After rechromatography of this material on a Vydac C₄ column (Fig. 1B), the immunoreactive BK was associated with the major peak (arrows). Further chromatography on a Vydac phenyl column (Fig. 1C) revealed that the material was extremely heterogeneous, and the immunoreactive BK was associated with the minor peak (arrows). Eel BK was purified to near homogeneity by a final chromatography on a Supelcosil C₁₈ column (Fig. 1D). The final yield of purified peptide was ~1 nmol.

Structural characterization. The primary structure of purified peptide was established as Arg(41)-Arg(49)-Pro(68)-Pro(71)-Gly(59)-Ser(15)-Trp(27)-Pro(40)-Leu(32)-Arg(19), where the values in parentheses are the yields of amino acid phenylthiohydantoins in picomoles. The proposed structure was confirmed by mass spectrometry, inasmuch as the observed molecular mass (1,220.9) was almost identical to the calculated monoisotopic molecular mass (1,220.7).

Physiological Studies

Experiment 1. Bolus injections of the eel BK-related peptides potently inhibited drinking in seawater eels (Fig. 2). The magnitude of the antidipsogenic effect was dose dependent, and significant differences were detected among different doses of [Arg⁰]BK. The duration of the antidipsogenic effect was longer at higher doses; the inhibition lasted for ~30 min after 10 nmol/kg of [Arg⁰]BK (Fig. 3). The BK peptides also induced an immediate increase in arterial pressure that was followed by a small but long-lasting increase (Fig. 2). In two of eight eels, the decrease in pressure between the two increases was below the basal level and so represented a triphasic increase-decrease-increase pattern, as reported in the trout after [Arg⁰]BK injection (20). The heart rate decreased immediately after injection and then slowly increased above the initial level (Fig. 2). Vehicle injection did not significantly alter drinking rate and cardiovascular parameters. Among the three BK peptides examined, [Arg⁰]BK was most potent followed by BK and [Arg⁰]-des-Arg⁹-BK, both of which had a similar potency for the antidipsogenic effect (Fig. 4). The dose dependency of the antidipsogenic effect was statistically significant (P < 0.05) only with [Arg⁰]BK. The lack of the dose dependency with BK and [Arg⁰]-des-Arg⁹-BK is due to the variability of the effects among individuals. However, BK was much more potent than [Arg⁰]-des-Arg⁹-BK for vascular and cardiac effects (Fig. 5), with [Arg⁰]BK being the most potent, as in the antidipsogenic effect. The relative potency of BK and [Arg⁰]-des-Arg⁹-BK was significantly different between antidipsogenic and cardiovascular (vasopressor or negative chronotropic) effects at 1 nmol/kg. The dose-response relationship was not apparent for the cardiovascular effects because of strong tachyphylaxis observed at high doses.

View larger version (45K): [in this window] [in a new window]   Fig. 2.   Drinking rate, dorsal aortic pressure, and heart rate in an eel recorded before and after injection of 10 nmol/kg of eel [Arg0]BK. Each spike on the water intake trace indicates 30 µl of water drunk as measured by the drop counter. Data from a single experiment are shown.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Time course of changes in water intake after injection of 10 nmol/kg of eel [Arg0]BK in seawater-adapted eels (n = 8). Values are means ± SE. *P < 0.05 compared with intake before injection. Arrow, point of injection.

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Dose-response relationship for the antidipsogenic effect of eel [Arg0]BK, BK, and [Arg0]-des-Arg9-BK in seawater-adapted eels (n = 8). Open bars, intake after injection of vehicle. Values are mean ± SE. *P < 0.05 compared with intake after vehicle injection as determined by Bonferroni/Dunn's test or compared between 2 different doses as determined by a randomization test for matched pairs (25). Regression line was linear only with [Arg0]BK.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Comparison of effects of eel [Arg0]BK, BK, and [Arg0]-des-Arg9-BK (1 nmol/kg) on drinking rate, dorsal aortic pressure, and heart rate in seawater-adapted eels (n = 8). Values are means ± SE. *Significantly different (P < 0.05) from [Arg0]BK. n.s., not significant.

Experiments 2 and 3. Slow infusion of [Arg⁰]BK inhibited drinking dose dependently (P < 0.05) between 10 and 1,000 pmol · kg¹ · min¹ in seawater-adapted eels. The decrease was significant at infusion rates of 100 and 1,000 pmol · kg¹ · min¹, although the plasma level of dipsogenic ANG II increased concomitantly (Fig. 6). The inhibition lasted for 30 min after termination of [Arg⁰]BK infusion. In contrast, arterial pressure did not increase significantly during [Arg⁰]BK infusion, despite a synergic action of vasoactive ANG II, the circulating concentration of which increased at high doses (Fig. 6). [Arg⁰]BK infusion did not alter plasma Na⁺ concentration and osmolality at any doses examined. Hematocrit decreased gradually during the course of infusions, and the decrease became significant (P < 0.05) during the last saline infusion. However, [Arg⁰]BK did not change hematocrit at any doses.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Effects of eel [Arg0]BK infusions on drinking rate, dorsal aortic pressure, and plasma ANG II concentration in seawater-adapted eels (n = 6). Values are means ± SE. *P < 0.05 compared with the value during the initial infusion of vehicle as determined by a Bonferroni/Dunn's test or compared between 2 different doses as determined by a randomization test for matched pairs (25). Regression line of the dose-response curve was linear between 10 and 1,000 pmol · kg1 · min1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the present study, a BK-related peptide (eel [Arg⁰]BK) was isolated from kallikrein-treated plasma of the Japanese eel A. japonica, and its amino acid sequence was determined. Using a synthetic replicate of the eel [Arg⁰]BK, novel and potent antidipsogenic effects were demonstrated in seawater-adapted eels that drink continuously at high rates. Bolus injections of eel [Arg⁰]BK also exhibited biphasic vasopressor and cardiac effects that were similar to those demonstrated in other teleost species (19, 20). Slow infusions of eel [Arg⁰]BK inhibited drinking without changes in arterial pressure, confirming its potent antidipsogenic effect.

Biochemical Studies

As well as being the most potent with respect to cardiovascular and antidipsogenic effects in the eel, [Arg⁰]BK is appreciably more potent than BK in its vasopressor action in the cod in vivo (20) and in its contractile action on trout gastric smooth muscle in vitro (13). This suggests that the endogenous active peptide circulating in the blood in teleost fish may be [Arg⁰]BK rather than BK. Unfortunately, the antiserum to mammalian BK used in this study was not sufficiently sensitive to detect circulating levels of BK in the eel arising from activation of the animal's kallikrein-kinin system. To determine the circulating form of BK and the changes in the plasma concentration after physiological stimuli in fish, it is necessary to develop a sensitive and specific radioimmunoassay for [Arg⁰,Trp⁵,Leu⁸]BK for teleost fishes.

In general, the primary structures of the BK-related peptides have been poorly conserved during evolution, and the variation in amino acids occurs at positions 1, 2, 5, 6, 7, and 8. It is somewhat surprising, therefore, that teleost species (eel, trout, and cod) that are only distantly related and arose from ancient divergences (18) have an identical BK sequence. In other oligopeptide hormones similar in size to BK such as ANG I, 7 of 10 amino acid residues are conserved across different vertebrate groups, but trout and eel have different sequences (27). Consequently, the fact that the sequence of BK has been strongly conserved in teleost fish may indicate its essential physiological functions in this vertebrate group.

Physiological Studies

In mammals such as the rat (11), BK is recognized as a vasoactive hormone that causes an immediate hypotension when injected systemically through a decrease in peripheral resistance (6, 21, 23). BK also exhibits a weak dipsogenic effect when injected systemically with captopril (8). Therefore, the vasopressor and antidipsogenic effects of native BK peptides in the eel are largely opposite to the effects observed in mammals. In other nonmammalian species, the effects of BK on drinking have not been examined, but homologous BK has diverse cardiovascular actions in birds (14) and reptiles (2, 4, 34). In vitro, BK is vasorelaxant in isolated arteries of mammals, but it is only weakly vasoactive in the isolated vessels and the perfused trunk of the trout (5).

In the present study, bolus injections and slow infusions of [Arg⁰]BK, the latter mimicking more closely the physiological changes that may occur in vivo, inhibited drinking in seawater eels. The inhibition occurred even though plasma ANG II concentration increased to the level strongly dipsogenic in the eel (33). Therefore, it is obvious that [Arg⁰]BK is a strong antidipsogenic hormone in the eel. The increase in plasma ANG II concentration caused by BK did not increase arterial pressure in the present study, although the similar increase of ANG II alone caused by infusion was vasopressor (33). It seems that interactions of multiple hormonal systems, including the renin-angiotensin and kallikrein-kinin systems, should have occurred for physiological regulation of body fluid and pressure homeostasis in the eel.

Our previous study showed that infusion of atrial natriuretic peptide (ANP) decreases plasma Na⁺ concentration with concomitant inhibition of drinking in seawater eels (29). In the present study, BK did not alter plasma Na⁺ concentration and osmolality, even though drinking was inhibited to the level similar to that of ANP inhibition. Thus it seems that the decrease in plasma Na⁺ concentration after ANP is due to its potent inhibitory action on intestinal NaCl absorption (16), while BK may not act on the intestine to inhibit NaCl absorption in the eel. Because hematocrit also did not change during BK infusions, BK may not have altered blood volume, although hematocrit is not an ideal marker for changes in blood volume in the eel (26).

Perspectives

The site of antidipsogenic action of eel [Arg⁰]BK was not addressed in this study, but it is possible that the peptide acts on the brain to modulate drinking rate as observed for the dipsogenic effect of ANG II in the same species (28). A precise regulation of the rate of drinking is essential to the survival of marine teleosts that exist in a hyperosmotic environment. Our observation that infusion of high doses of [Arg⁰]BK causes the release of ANG II suggests that the kallikrein-kinin and renin-angiotensin systems may operate in opposition to regulate the rate of drinking in the teleost fish. An integrative approach to the interaction of multiple hormonal systems for regulation of drinking may assume increasing importance in the future.