Fish Physiology and Biochemistry vol. 11 no. 1-6 pp 183-188 (1993) Kugler Publications, Amsterdam/New York
Biochemistry and physiology of a family of eel natriuretic peptides Yoshio Takeil and Richard J. Balment 2 'Laboratory of Physiology, Ocean Research Institute, University of Tokyo, Nakano, Tokyo 164, Japan; 2 Department of Physiological Sciences, University of Manchester, Manchester M13 9PT, U.K.
Keywords: atrial natriuretic peptide, brain natriuretic peptide, C-type natriuretic peptide, ventricular natriuretic peptide, Anguilla japonica, amino acid sequence, hydromineral balance, water intake, intestinal absorption, renal excretion
Abstract Three natriuretic peptides with similar structures were isolated from eels and their amino acid sequences determined; atrial natriuretic peptide (ANP) from atria, ventricular natriuretic peptide (VNP) from ventricles, and C-type natriuretic peptide (CNP) from brains. All three hormones were circulating in eel blood, and their plasma levels invariably decreased when eels were transferred from fresh water (FW) to seawater (SW). Eel ANP and VNP inhibited drinking in FW and SW eels. Eel ANP inhibited water and Na + absorption by the intestine of SW eels. The potency of these ANP effects was 2-3 orders greater than those of other hormones which are known to have similar effects. Eel ANP and VNP induced antidiuresis but not antinatriuresis in FW eels. Eel ANP increased plasma cortisol level in SW eels but not in FW eels. The antidiuretic effect and the stimulation of cortisol secretion in eels are opposite to the ANP effects reported in mammals. These data suggest that ANP plays a complex role in the eel osmoregulation.
Resume Trois peptides natriuretiques pr6sentant des structures similaires ont te isol6s chez l'anguille et leurs squences en acides amin6s a t6 d6termin6es; le peptide atrial natriur6tique (ANP) dans l'atria, le peptide ventriculaire natriur6tique (VNP) dans les ventricules et le peptide natriur6tique de type C dans le cerveau. Ces trois hormones sont presents dans le sang d'anguille et leur niveau plasmatique decroit invariablement quand les anguilles sont transf6r6es d'eau douce en eau de mer. L'ANP et le VNP d'anguille inhibent l'action de boire chez les anguilles d'eau douce et d'eau de mer. L'ANP d'anguille inhibe l'absorption d'eau et de Na + par l'intestin d'anguille en eau de mer. Ces effets de I'ANP sont 2 3 fois plus grands que ceux observes avec d'autres hormones qui sont connues pour avoir des effets similaires. L'ANP et le VNP d'anguille induisent l'antidiurese mais pas l'antinatriurese chez les anguilles d'eau douce. L'ANP d'anguille augmente chez ce poisson les niveaux de cortisol plasmatique en eau de mer mais pas en eau douce. L'effet antidiur6tique et la stimulation de la scr6tion de cortisol chez l'anguille sont contraire aux effets de I'ANP observes chez les mammiferes. Ces rsultats suggerent que I'ANP joue un role complexe dans l'osmoregulation de l'anguille. Correspondence to: Dr. Yoshio Takei, Laboratory of Physiology, Ocean Research Institute, University of Tokyo, 1-15-1 Minamidai, Nakano, Tokyo 164, Japan
184 Biochemistry of eel ANP
Atrial (A-type) natriuretic peptide Eel Bullfrog Man 2 Rat
F K L R RF F RIS RI
SK
SLR SLR
RI
S Y
Brain (B-type) natriuretic peptide Fowl Pig Dog Cattle Man
SPK SPK PK SPK
Rat
S-K
IMIRDG- RR G S GSKN -RD G-FRR SGLGNVRY G R G VLKY RR GS LGLGNVLRY RG -F RKDR SS GLKVLRH VQG H
S-
QKIR
A
R
DG
F
C-type natriuretic peptide 3
Dogfish Eel Killifish Bullfrog Fowl Man
PSRG CF LDRI GLG GLG NRG- F LDRI LG GNRG- F LDRIG L G CFFSRGLDRI GSRS- CFKLRI SMGLG SKG RI ventricular natriur..etic peptide..........
Ventricular natriuretic peptide Eel
KSFNS-CFGTRMDRIGSWSGLGCNSL-KNGTKKKIFGN o..................
Fig. 1. Comparison of amino acid sequences of natriuretic peptides from various vertebrate classes. Gaps are introduced to optimize matching. Identical sequences in each group across differ2 ent species are boxed. Dog, pig, and ox; mouse and rabbit; 4 3 Scyliorhinus canicula and Triakis scyllia; and rat and pig; share identical sequences.
Introduction For terrestrial animals, retention of water and Na +, i.e., conservation of blood volume, is essential for their survival. It is perhaps not surprising, therefore, that atrial natriuretic peptide (ANP), a hormone that promotes water and Na + loss, was not discovered until the last decade (de Bold et al. 1981). For aquatic animals like fishes, however, they have to extrude water in FW and to extrude Na + in SW. Thus, ANP may have more important roles in fishes than in terrestrial animals if it also promotes water and Na + loss in fishes. It should also be noted that in fishes, water and Na+ movement are in opposite directions, i.e., water is excreted while Na + is retained in FW fishes. Thus, fishes may provide good models to examine the effects of ANP, as the effects on water and Na + are potentially separable in this group.
Since the initial discovery of ANP in mammalian atria, B-type (brain) natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were identified in the heart and brain, respectively (Fig. 1). Thus, it is clear that ANP is not a single peptide but part of a family of peptides with similar structures. ANP was isolated from eel atria and its amino acid sequence determined (Takei et al. 1989). Eel ANP has a sequence homology of 62% to mammalian ANP. We recently determined the cDNA sequence of eel ANP (Ueki M. and Takei Y., unpublished data), which revealed the presence of a glycine residue just after the C-terminal lysine residue. We are now examining whether the glycine residue is used for amidation of the C-terminal amino acid or whether it exists, in fact, at the C-terminus. Fish BNP has not been identified yet. CNP was isolated from the killifish and eel brains (Price et al. 1990; Takei et al. 1990a). A CNP prohormone was also isolated from the heart of two species of dogfish in addition to the mature CNP from the brain (Suzuki et al. 1991, 1992). The sequence identity of brain CNP was more than 80% even between mammals and sharks. A possibly new natriuretic peptide was isolated from the eel cardiac ventricle, and named ventricular natriuretic peptide (VNP) (Fig. 1; Takei et al. 1991). This peptide has a uniquely long Cterminal extension from the disulfide ring. Since the BNP sequence is highly variable, and since BNP has not been found in the eel, it is likely that VNP belongs to the BNP family. However, the cDNA of eel VNP lacks the seven repetition of ATTTA sequence in the 3' non-coding region (Takei Y. and Nishizawa T., unpublished data). The presence of the repetitive sequence is a characteristic of all BNP cDNAs so far sequenced (Akizuki et al. 1991). It remains to be determined whether the fourth natriuretic peptide, namely BNP, is present in the eel or not.
Natriureticpeptides in FW and SW eels Since eel ANP, VNP and CNP have more than 60% sequence identity, it is of the utmost importance to
185 Table 1. Natriuretic peptide concentration in tissue and plasma of FW eels and those adapted in SW for 2 weeks. Natriuretic peptides Tissue
ANP
VNP
CNP
Atrium (fmol/mg)
FW: SW:
10627+ 1647 56101+ 10283
2544 +310 3444 ±502
7+2 3 +1
Ventricle (fmol/mg)
FW: SW:
56±8 41 ± 13
1397 +±83 1265 ± 94
6+1 4+1
Brain (fmol/mg)
FW: SW:
22+1 30+3
27 + 14 3+±1
37±4 36 4
Plasma (fmol/ml)
FW: SW:
247 + 66 123 + 21
754 + 49 106± 11
468 + 90 28 + 10
Data are shown as means
SEM (n = 8)
establish a specific radioimmunoassay for each peptide; the antibody for one peptide should not cross-react with the other two. Using such specific radioimmunoassays, we measured ANP, VNP and CNP levels in the plasma, atrium, ventricle and brain of FW eels (Table 1). It is clear that ANP is an atrial peptide and CNP is a brain peptide. The VNP content of the ventricle was the greatest because of its large size, though atrial concentration was higher. Although ANP is a major circulating natriuretic peptide in mammals, plasma VNP levels are 3-fold higher and plasma CNP levels 2-fold higher than the ANP in FW eels (Table 1). The high plasma VNP level observed may be accounted for by our preliminary observation that VNP is continuously secreted from both atria and ventricles of eels (Takei Y. and Suzuki R., unpublished data). The high plasma concentration of CNP suggests that there must be a source of plasma CNP other than the brain in eels. The atrial ANP level was increased, and plasma ANP, VNP and CNP levels were all decreased after eels were transferred to SW for two weeks. The decrease in plasma peptide concentration was most profound for CNP, followed by VNP and ANP. Previous studies employing a radioimmunoassay for mammalian ANP showed that plasma ANPlike immunoreactivity was invariably increased after FW fishes were transferred to hypertonic media and decreased after SW fishes were transferred to hypotonic media (Westenfelder et al. 1988; Evans et al. 1989; Smith et al. 1991; see also Evans 1990;
Takei and Balment 1992 for reviews). It should be noted, however, the radioimmunoassay for human ANP which we used in previous studies measured not eel ANP but eel VNP (Takei et al. 1992). It is not yet clear whether the fall in plasma ANP, VNP and CNP on transfer of FW eels to SW is characteristic only of the eel. Specific radioimmunoassays for other fish species are not yet available. The changes in plasma ANP, VNP and CNP levels in response to salinity challenge do indicate, however, that the natriuretic peptides may be involved in fish osmoregulation.
Natriureticpeptides and osmoregulation in eels In terrestrial animals, regulation of body fluid water and Na + is achieved principally by balancing the oral and intestinal uptake and the renal excretion. In mammals, ANP inhibits both oral intake and intestinal absorption of water and Na+, and stimulates their excretion by the kidney (see Brenner et al. 1990). In addition, ANP inhibits the secretion of vasopressin and aldosterone. All of these effects act in concert to decrease blood volume. As a first step to assess the role of ANP in eel osmoregulation, therefore, it seems appropriate to examine the effect of eel natriuretic peptides on drinking, intestinal absorption, renal excretion, and secretion of cortisol, an inferred fish mineralocorticoid. Eel ANP inhibited drinking in oesophagealcannulated FW eels in a dose-dependent manner
186
eVNP 0.03nrno1/kg s_ e saline
eANP 0.03nmol/ka
-- ---------------
-hl~i l: 15 min
i-i- FFl
f
eVNP 0.1nmol/kg
---l-I-I
i l
t
eAVP O.lnmol/kg
A;==,;,
eANG eANG 1110nmol/kg _-E__
-7Lol
eVNP 0.3nmol/kg
-- 1
iX
eANP 0.3nmol/kg
Fig. 2. Changes in drinking rate after intraarterial injection of eel ANP, VNP and angiotensin II in an oesophageal-cannulated freshwater eel. Each spike indicates 0.03 ml of water drunk.
(Fig. 2). Eel VNP appeared to be more potent than eel ANP. The minimum effective dose was as little 40O as 10 pmol/kg, which was less than 1/100 of the I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ dose of eel angiotensin II required to stimulate i i _ I_ i~ ~~~~~~~~~ IiI drinking in FW eels (Fig. 2; Takei et al. 1979). Eel 20 i , ANP and VNP also inhibited the vigorous drinking of SW-exposed eels in a similar manner. Eel ANP inhibited Na + absorption by the intes-600 ANP c o tine of SW-adapted eels in a dose-dependent man-0.2 nmolkg o 400 . F (n=7) ner (Ando et al. 1992). Eel ANP also inhibited gut 2.0 nmolkg water absorption. Eel ANP was 100-fold more po. 200 tent than rat and human ANP, and was more po0) tent than all other inhibitory substances thus far D 0 reported such as serotonin and acetylcholine. Similar inhibitory effects were also reported in the 600 VNP flounder, Pseudopleuronectes americanus, using 0.2 nmoVkg 400 F (nEi) rat ANP (O'Grady 1989). / 2.0 (riM)V~ nmol/kg In contrast to the diuretic action described in 200 mammals, eel ANP and VNP produced an antidiuresis in FW eels (Fig. 3). The antidiuretic effect was 0 not observed at 0.2 nmol/kg of eel ANP, but it be-1-0 0-1 1-2 came evident at 2 nmol/kg. A clear antidiuresis was Post-injection (h) induced at 0.2 nmol/kg of eel VNP. There were no Fig. 3. Changes in urine production after intraarterial injection significant changes in Na + excretion at any dose of of eel ANP or VNP in freshwater eels. The bar in each column ANP or VNP despite the decreased urine flow rate. represents standard error of the mean. *p < 0.05 compared with pre-injection control values determined by Student's t-test. In the rainbow trout, Oncorhynchus mykiss and 600
vehicle (r8a)
187 toadfish, Opsanus tau, mammalian ANP or homologous heart extract apparently causes diuresis and natriuresis (Duff and Olson 1986; Lee and Malvin 1987). Plasma cortisol concentration increased after intraarterial injection of 0.2 nmol/kg of eel ANP in SW-adapted eels compared with vehicle-injected controls. Eel VNP was without effect. Interestingly, similar eel ANP administration failed to increase the cortisol level in FW eels. These results are comparable with those obtained in the flounder, Platichthysflesus, after injection of human ANP (Arnold-Reed and Balment 1991). Since aldosterone and cortisol secretion are inhibited by ANP in humans (Naruse et al. 1987), these results are again contrary to those observed in mammals. Although more detailed examination is necessary, each of the eel natriuretic peptides appears to be involved in the osmoregulation of eels. However, the action of these peptides in the eel appear to differ markedly from those reported in tetrapods.
Acknowledgements This investigation was supported in part by Grantin-Aid from the Ministry of Education, Science and Culture of Japan, the Royal Society and the Japan Society for the Promotion of Science.
References cited Akizuki, N., Kangawa, K., Minamino, N. and Matsuo, H. 1991. Cloning and sequence analysis of complementary DNA encoding a precursor for chicken natriuretic peptide. FEBS Lett. 280: 257-262. Ando, M., Kondo, K. and Takei, Y. 1992. Effects of eel atrial natriuretic peptide on NaCI and water transport across the intestine of the seawater eel. J. Comp. Physiol. B. 162: 436-439. Arnold-Reed, D.E. and Balment, R.J. 1991. Atrial natriuretic factor stimulates in vivo and in vitro secretion of cortisol in teleosts. J. Endocrinol. 128: R17-R20. Brenner, B.M., Ballerman, B.J., Gunning, M.E. and Zeidel, M.L. 1990. Diverse biological actions of atrial natriuretic peptide. Physiol. Rev. 70: 665-699. De Bold, A.J., Borenstein, H.B., Veress, A.T. and Sonnenberg, H. 1981. A rapid and potent natriuretic response to in-
travenous injection of atrial myocardial extract in rats. Life Sci. 28: 89-94. Duff, D.W. and Olson, K.R. 1986. Trout vascular and renal responses to atrial natriuretic factor and heart extract. Am. J. Physiol. 251: R639-R642. Evans, D.H. 1990. An emerging role for a cardiac peptide hormone in fish osmoregulation. Ann. Rev. Physiol. 52: 43-60. Evans, D.H., Chipouras, E. and Payne, J.A. 1989. Immunoreactive atriopeptin in plasma of fishes: its potential role in gill hemodynamics. Am. J. Physiol. 257: R939-R945. Lee, J. and Malvin, R.L. 1987. Natriuretic response to homologous heart extract in aglomerular toadfish. Am. J. Physiol. 252: R1055-R1058. Naruse, M., Obata, K., Naruse, K., Yamaguchi, H., Demura, H., Inagami, T. and Shizume, K. 1987. Atrial natriuretic polypeptide inhibits cortisol secretion as well as aldosterone secretion in vitro from human adrenal tissue. J. Clin. Endocrinol. Metab. 64: 10-16. O'Grady, S.M. 1989. Cyclic nucleotide-mediated effects of ANF and VIP on flounder intestinal ion transport. Am. J. Physiol. 256: C142-C146. Price, D.A., Doble, K.E., Lee, T.D., Galli, S.M., Dunn, B.M., Parten, B. and Evans, D.H. 1990. The sequencing, synthesis, and biological actions of an ANP-like peptide isolated from the brain of killifish Fundulus heteroclitus. Biol. Bull. 178: 279-285. Smith, N.F., Eddy, F.B., Struthers, A.D. and Talbot, C. 1991. Renin, atrial natriuretic peptide and blood plasma ions in parr and smolts of Atlantic salmon Salmo salar L. and rainbow trout Oncorhynchus mykiss (Walbaum) in fresh water and after short-term exposure to sea water. J. Exp. Biol. 157: 63-74. Suzuki, R., Takahashi, A., Hazon, N. and Takei, Y. 1991. Isolation of high-molecular-weight C-type natriuretic peptide from the heart of a cartilaginous fish (European dogfish, Scyliorhinus canicula). FEBS Lett. 282: 321-325. Suzuki, R., Takahashi, A. and Takei, Y. 1992. Different molecular forms of C-type natriuretic peptide isolated from the brain and heart of an elasmobranch, Triakisscyllia. J. Endocrinol. 135: 317-323. Takei, Y., Ando, K., and Kawakami, M. 1992. Atrial natriuretic peptide in eel plasma, heart and brain characterized by homologous radioimmunoassay. J. Endocrinol. 135: 325331. Takei, Y. and Balment, R.J. 1992. Natriuretic factors in nonmammalian vertebrates. In New Insights into Vertebrate Kidney Function. Edited by J.A. Brown, R.J. Balment and J.C. Rankin. Cambridge University Press, Cambridge. 351-385. Takei, Y., Hirano, T. and Kobayashi, H. 1979. Angiotensin and water intake in the eel, Anguilla japonica. Gen. Comp. Endocrinol. 38: 466-475. Takei, Y., Takahashi, A., Watanabe, T.X., Nakajima, K. and Sakakibara, S. 1989. Amino acid sequence and relative biological activity of eel atrial natriuretic peptide. Biochem. Biophys. Res. Commun. 164: 537-543. Takei, Y., Takahashi, A., Watanabe, T.X., Nakajima, K. and Sakakibara, S. 1991. A novel natriuretic peptide isolated from eel cardiac ventricles. FEBS Lett. 282: 317-320.
188 Takei, Y., Takahashi, A., Watanabe, T.X., Nakajima, K., Sakakibara, S., Takao, T. and Shimonishi, Y. 1990a. Amino acid sequence and relative biological activity of a natriuretic peptide isolated from eel brain. Biochem. Biophys. Res. Commun. 170: 883-891.
Westenfelder, C., Birch, F.M., Baranowski, R.L., Rosenfeld, M.J., Shiozawa, D.K. and Kablitz, C. 1988. Atrial natriuretic factor and salt adaptation in the teleost fish Gala atraria. Am. J. Physiol. 255: F1281-1286.
Biochemistry and physiology of a family of eel natriuretic peptides Yoshio Takeil and Richard J. Balment 2 'Laboratory of Physiology, Ocean Research Institute, University of Tokyo, Nakano, Tokyo 164, Japan; 2 Department of Physiological Sciences, University of Manchester, Manchester M13 9PT, U.K.
Keywords: atrial natriuretic peptide, brain natriuretic peptide, C-type natriuretic peptide, ventricular natriuretic peptide, Anguilla japonica, amino acid sequence, hydromineral balance, water intake, intestinal absorption, renal excretion
Abstract Three natriuretic peptides with similar structures were isolated from eels and their amino acid sequences determined; atrial natriuretic peptide (ANP) from atria, ventricular natriuretic peptide (VNP) from ventricles, and C-type natriuretic peptide (CNP) from brains. All three hormones were circulating in eel blood, and their plasma levels invariably decreased when eels were transferred from fresh water (FW) to seawater (SW). Eel ANP and VNP inhibited drinking in FW and SW eels. Eel ANP inhibited water and Na + absorption by the intestine of SW eels. The potency of these ANP effects was 2-3 orders greater than those of other hormones which are known to have similar effects. Eel ANP and VNP induced antidiuresis but not antinatriuresis in FW eels. Eel ANP increased plasma cortisol level in SW eels but not in FW eels. The antidiuretic effect and the stimulation of cortisol secretion in eels are opposite to the ANP effects reported in mammals. These data suggest that ANP plays a complex role in the eel osmoregulation.
Resume Trois peptides natriuretiques pr6sentant des structures similaires ont te isol6s chez l'anguille et leurs squences en acides amin6s a t6 d6termin6es; le peptide atrial natriur6tique (ANP) dans l'atria, le peptide ventriculaire natriur6tique (VNP) dans les ventricules et le peptide natriur6tique de type C dans le cerveau. Ces trois hormones sont presents dans le sang d'anguille et leur niveau plasmatique decroit invariablement quand les anguilles sont transf6r6es d'eau douce en eau de mer. L'ANP et le VNP d'anguille inhibent l'action de boire chez les anguilles d'eau douce et d'eau de mer. L'ANP d'anguille inhibe l'absorption d'eau et de Na + par l'intestin d'anguille en eau de mer. Ces effets de I'ANP sont 2 3 fois plus grands que ceux observes avec d'autres hormones qui sont connues pour avoir des effets similaires. L'ANP et le VNP d'anguille induisent l'antidiurese mais pas l'antinatriurese chez les anguilles d'eau douce. L'ANP d'anguille augmente chez ce poisson les niveaux de cortisol plasmatique en eau de mer mais pas en eau douce. L'effet antidiur6tique et la stimulation de la scr6tion de cortisol chez l'anguille sont contraire aux effets de I'ANP observes chez les mammiferes. Ces rsultats suggerent que I'ANP joue un role complexe dans l'osmoregulation de l'anguille. Correspondence to: Dr. Yoshio Takei, Laboratory of Physiology, Ocean Research Institute, University of Tokyo, 1-15-1 Minamidai, Nakano, Tokyo 164, Japan
184 Biochemistry of eel ANP
Atrial (A-type) natriuretic peptide Eel Bullfrog Man 2 Rat
F K L R RF F RIS RI
SK
SLR SLR
RI
S Y
Brain (B-type) natriuretic peptide Fowl Pig Dog Cattle Man
SPK SPK PK SPK
Rat
S-K
IMIRDG- RR G S GSKN -RD G-FRR SGLGNVRY G R G VLKY RR GS LGLGNVLRY RG -F RKDR SS GLKVLRH VQG H
S-
QKIR
A
R
DG
F
C-type natriuretic peptide 3
Dogfish Eel Killifish Bullfrog Fowl Man
PSRG CF LDRI GLG GLG NRG- F LDRI LG GNRG- F LDRIG L G CFFSRGLDRI GSRS- CFKLRI SMGLG SKG RI ventricular natriur..etic peptide..........
Ventricular natriuretic peptide Eel
KSFNS-CFGTRMDRIGSWSGLGCNSL-KNGTKKKIFGN o..................
Fig. 1. Comparison of amino acid sequences of natriuretic peptides from various vertebrate classes. Gaps are introduced to optimize matching. Identical sequences in each group across differ2 ent species are boxed. Dog, pig, and ox; mouse and rabbit; 4 3 Scyliorhinus canicula and Triakis scyllia; and rat and pig; share identical sequences.
Introduction For terrestrial animals, retention of water and Na +, i.e., conservation of blood volume, is essential for their survival. It is perhaps not surprising, therefore, that atrial natriuretic peptide (ANP), a hormone that promotes water and Na + loss, was not discovered until the last decade (de Bold et al. 1981). For aquatic animals like fishes, however, they have to extrude water in FW and to extrude Na + in SW. Thus, ANP may have more important roles in fishes than in terrestrial animals if it also promotes water and Na + loss in fishes. It should also be noted that in fishes, water and Na+ movement are in opposite directions, i.e., water is excreted while Na + is retained in FW fishes. Thus, fishes may provide good models to examine the effects of ANP, as the effects on water and Na + are potentially separable in this group.
Since the initial discovery of ANP in mammalian atria, B-type (brain) natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) were identified in the heart and brain, respectively (Fig. 1). Thus, it is clear that ANP is not a single peptide but part of a family of peptides with similar structures. ANP was isolated from eel atria and its amino acid sequence determined (Takei et al. 1989). Eel ANP has a sequence homology of 62% to mammalian ANP. We recently determined the cDNA sequence of eel ANP (Ueki M. and Takei Y., unpublished data), which revealed the presence of a glycine residue just after the C-terminal lysine residue. We are now examining whether the glycine residue is used for amidation of the C-terminal amino acid or whether it exists, in fact, at the C-terminus. Fish BNP has not been identified yet. CNP was isolated from the killifish and eel brains (Price et al. 1990; Takei et al. 1990a). A CNP prohormone was also isolated from the heart of two species of dogfish in addition to the mature CNP from the brain (Suzuki et al. 1991, 1992). The sequence identity of brain CNP was more than 80% even between mammals and sharks. A possibly new natriuretic peptide was isolated from the eel cardiac ventricle, and named ventricular natriuretic peptide (VNP) (Fig. 1; Takei et al. 1991). This peptide has a uniquely long Cterminal extension from the disulfide ring. Since the BNP sequence is highly variable, and since BNP has not been found in the eel, it is likely that VNP belongs to the BNP family. However, the cDNA of eel VNP lacks the seven repetition of ATTTA sequence in the 3' non-coding region (Takei Y. and Nishizawa T., unpublished data). The presence of the repetitive sequence is a characteristic of all BNP cDNAs so far sequenced (Akizuki et al. 1991). It remains to be determined whether the fourth natriuretic peptide, namely BNP, is present in the eel or not.
Natriureticpeptides in FW and SW eels Since eel ANP, VNP and CNP have more than 60% sequence identity, it is of the utmost importance to
185 Table 1. Natriuretic peptide concentration in tissue and plasma of FW eels and those adapted in SW for 2 weeks. Natriuretic peptides Tissue
ANP
VNP
CNP
Atrium (fmol/mg)
FW: SW:
10627+ 1647 56101+ 10283
2544 +310 3444 ±502
7+2 3 +1
Ventricle (fmol/mg)
FW: SW:
56±8 41 ± 13
1397 +±83 1265 ± 94
6+1 4+1
Brain (fmol/mg)
FW: SW:
22+1 30+3
27 + 14 3+±1
37±4 36 4
Plasma (fmol/ml)
FW: SW:
247 + 66 123 + 21
754 + 49 106± 11
468 + 90 28 + 10
Data are shown as means
SEM (n = 8)
establish a specific radioimmunoassay for each peptide; the antibody for one peptide should not cross-react with the other two. Using such specific radioimmunoassays, we measured ANP, VNP and CNP levels in the plasma, atrium, ventricle and brain of FW eels (Table 1). It is clear that ANP is an atrial peptide and CNP is a brain peptide. The VNP content of the ventricle was the greatest because of its large size, though atrial concentration was higher. Although ANP is a major circulating natriuretic peptide in mammals, plasma VNP levels are 3-fold higher and plasma CNP levels 2-fold higher than the ANP in FW eels (Table 1). The high plasma VNP level observed may be accounted for by our preliminary observation that VNP is continuously secreted from both atria and ventricles of eels (Takei Y. and Suzuki R., unpublished data). The high plasma concentration of CNP suggests that there must be a source of plasma CNP other than the brain in eels. The atrial ANP level was increased, and plasma ANP, VNP and CNP levels were all decreased after eels were transferred to SW for two weeks. The decrease in plasma peptide concentration was most profound for CNP, followed by VNP and ANP. Previous studies employing a radioimmunoassay for mammalian ANP showed that plasma ANPlike immunoreactivity was invariably increased after FW fishes were transferred to hypertonic media and decreased after SW fishes were transferred to hypotonic media (Westenfelder et al. 1988; Evans et al. 1989; Smith et al. 1991; see also Evans 1990;
Takei and Balment 1992 for reviews). It should be noted, however, the radioimmunoassay for human ANP which we used in previous studies measured not eel ANP but eel VNP (Takei et al. 1992). It is not yet clear whether the fall in plasma ANP, VNP and CNP on transfer of FW eels to SW is characteristic only of the eel. Specific radioimmunoassays for other fish species are not yet available. The changes in plasma ANP, VNP and CNP levels in response to salinity challenge do indicate, however, that the natriuretic peptides may be involved in fish osmoregulation.
Natriureticpeptides and osmoregulation in eels In terrestrial animals, regulation of body fluid water and Na + is achieved principally by balancing the oral and intestinal uptake and the renal excretion. In mammals, ANP inhibits both oral intake and intestinal absorption of water and Na+, and stimulates their excretion by the kidney (see Brenner et al. 1990). In addition, ANP inhibits the secretion of vasopressin and aldosterone. All of these effects act in concert to decrease blood volume. As a first step to assess the role of ANP in eel osmoregulation, therefore, it seems appropriate to examine the effect of eel natriuretic peptides on drinking, intestinal absorption, renal excretion, and secretion of cortisol, an inferred fish mineralocorticoid. Eel ANP inhibited drinking in oesophagealcannulated FW eels in a dose-dependent manner
186
eVNP 0.03nrno1/kg s_ e saline
eANP 0.03nmol/ka
-- ---------------
-hl~i l: 15 min
i-i- FFl
f
eVNP 0.1nmol/kg
---l-I-I
i l
t
eAVP O.lnmol/kg
A;==,;,
eANG eANG 1110nmol/kg _-E__
-7Lol
eVNP 0.3nmol/kg
-- 1
iX
eANP 0.3nmol/kg
Fig. 2. Changes in drinking rate after intraarterial injection of eel ANP, VNP and angiotensin II in an oesophageal-cannulated freshwater eel. Each spike indicates 0.03 ml of water drunk.
(Fig. 2). Eel VNP appeared to be more potent than eel ANP. The minimum effective dose was as little 40O as 10 pmol/kg, which was less than 1/100 of the I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ dose of eel angiotensin II required to stimulate i i _ I_ i~ ~~~~~~~~~ IiI drinking in FW eels (Fig. 2; Takei et al. 1979). Eel 20 i , ANP and VNP also inhibited the vigorous drinking of SW-exposed eels in a similar manner. Eel ANP inhibited Na + absorption by the intes-600 ANP c o tine of SW-adapted eels in a dose-dependent man-0.2 nmolkg o 400 . F (n=7) ner (Ando et al. 1992). Eel ANP also inhibited gut 2.0 nmolkg water absorption. Eel ANP was 100-fold more po. 200 tent than rat and human ANP, and was more po0) tent than all other inhibitory substances thus far D 0 reported such as serotonin and acetylcholine. Similar inhibitory effects were also reported in the 600 VNP flounder, Pseudopleuronectes americanus, using 0.2 nmoVkg 400 F (nEi) rat ANP (O'Grady 1989). / 2.0 (riM)V~ nmol/kg In contrast to the diuretic action described in 200 mammals, eel ANP and VNP produced an antidiuresis in FW eels (Fig. 3). The antidiuretic effect was 0 not observed at 0.2 nmol/kg of eel ANP, but it be-1-0 0-1 1-2 came evident at 2 nmol/kg. A clear antidiuresis was Post-injection (h) induced at 0.2 nmol/kg of eel VNP. There were no Fig. 3. Changes in urine production after intraarterial injection significant changes in Na + excretion at any dose of of eel ANP or VNP in freshwater eels. The bar in each column ANP or VNP despite the decreased urine flow rate. represents standard error of the mean. *p < 0.05 compared with pre-injection control values determined by Student's t-test. In the rainbow trout, Oncorhynchus mykiss and 600
vehicle (r8a)
187 toadfish, Opsanus tau, mammalian ANP or homologous heart extract apparently causes diuresis and natriuresis (Duff and Olson 1986; Lee and Malvin 1987). Plasma cortisol concentration increased after intraarterial injection of 0.2 nmol/kg of eel ANP in SW-adapted eels compared with vehicle-injected controls. Eel VNP was without effect. Interestingly, similar eel ANP administration failed to increase the cortisol level in FW eels. These results are comparable with those obtained in the flounder, Platichthysflesus, after injection of human ANP (Arnold-Reed and Balment 1991). Since aldosterone and cortisol secretion are inhibited by ANP in humans (Naruse et al. 1987), these results are again contrary to those observed in mammals. Although more detailed examination is necessary, each of the eel natriuretic peptides appears to be involved in the osmoregulation of eels. However, the action of these peptides in the eel appear to differ markedly from those reported in tetrapods.
Acknowledgements This investigation was supported in part by Grantin-Aid from the Ministry of Education, Science and Culture of Japan, the Royal Society and the Japan Society for the Promotion of Science.
References cited Akizuki, N., Kangawa, K., Minamino, N. and Matsuo, H. 1991. Cloning and sequence analysis of complementary DNA encoding a precursor for chicken natriuretic peptide. FEBS Lett. 280: 257-262. Ando, M., Kondo, K. and Takei, Y. 1992. Effects of eel atrial natriuretic peptide on NaCI and water transport across the intestine of the seawater eel. J. Comp. Physiol. B. 162: 436-439. Arnold-Reed, D.E. and Balment, R.J. 1991. Atrial natriuretic factor stimulates in vivo and in vitro secretion of cortisol in teleosts. J. Endocrinol. 128: R17-R20. Brenner, B.M., Ballerman, B.J., Gunning, M.E. and Zeidel, M.L. 1990. Diverse biological actions of atrial natriuretic peptide. Physiol. Rev. 70: 665-699. De Bold, A.J., Borenstein, H.B., Veress, A.T. and Sonnenberg, H. 1981. A rapid and potent natriuretic response to in-
travenous injection of atrial myocardial extract in rats. Life Sci. 28: 89-94. Duff, D.W. and Olson, K.R. 1986. Trout vascular and renal responses to atrial natriuretic factor and heart extract. Am. J. Physiol. 251: R639-R642. Evans, D.H. 1990. An emerging role for a cardiac peptide hormone in fish osmoregulation. Ann. Rev. Physiol. 52: 43-60. Evans, D.H., Chipouras, E. and Payne, J.A. 1989. Immunoreactive atriopeptin in plasma of fishes: its potential role in gill hemodynamics. Am. J. Physiol. 257: R939-R945. Lee, J. and Malvin, R.L. 1987. Natriuretic response to homologous heart extract in aglomerular toadfish. Am. J. Physiol. 252: R1055-R1058. Naruse, M., Obata, K., Naruse, K., Yamaguchi, H., Demura, H., Inagami, T. and Shizume, K. 1987. Atrial natriuretic polypeptide inhibits cortisol secretion as well as aldosterone secretion in vitro from human adrenal tissue. J. Clin. Endocrinol. Metab. 64: 10-16. O'Grady, S.M. 1989. Cyclic nucleotide-mediated effects of ANF and VIP on flounder intestinal ion transport. Am. J. Physiol. 256: C142-C146. Price, D.A., Doble, K.E., Lee, T.D., Galli, S.M., Dunn, B.M., Parten, B. and Evans, D.H. 1990. The sequencing, synthesis, and biological actions of an ANP-like peptide isolated from the brain of killifish Fundulus heteroclitus. Biol. Bull. 178: 279-285. Smith, N.F., Eddy, F.B., Struthers, A.D. and Talbot, C. 1991. Renin, atrial natriuretic peptide and blood plasma ions in parr and smolts of Atlantic salmon Salmo salar L. and rainbow trout Oncorhynchus mykiss (Walbaum) in fresh water and after short-term exposure to sea water. J. Exp. Biol. 157: 63-74. Suzuki, R., Takahashi, A., Hazon, N. and Takei, Y. 1991. Isolation of high-molecular-weight C-type natriuretic peptide from the heart of a cartilaginous fish (European dogfish, Scyliorhinus canicula). FEBS Lett. 282: 321-325. Suzuki, R., Takahashi, A. and Takei, Y. 1992. Different molecular forms of C-type natriuretic peptide isolated from the brain and heart of an elasmobranch, Triakisscyllia. J. Endocrinol. 135: 317-323. Takei, Y., Ando, K., and Kawakami, M. 1992. Atrial natriuretic peptide in eel plasma, heart and brain characterized by homologous radioimmunoassay. J. Endocrinol. 135: 325331. Takei, Y. and Balment, R.J. 1992. Natriuretic factors in nonmammalian vertebrates. In New Insights into Vertebrate Kidney Function. Edited by J.A. Brown, R.J. Balment and J.C. Rankin. Cambridge University Press, Cambridge. 351-385. Takei, Y., Hirano, T. and Kobayashi, H. 1979. Angiotensin and water intake in the eel, Anguilla japonica. Gen. Comp. Endocrinol. 38: 466-475. Takei, Y., Takahashi, A., Watanabe, T.X., Nakajima, K. and Sakakibara, S. 1989. Amino acid sequence and relative biological activity of eel atrial natriuretic peptide. Biochem. Biophys. Res. Commun. 164: 537-543. Takei, Y., Takahashi, A., Watanabe, T.X., Nakajima, K. and Sakakibara, S. 1991. A novel natriuretic peptide isolated from eel cardiac ventricles. FEBS Lett. 282: 317-320.
188 Takei, Y., Takahashi, A., Watanabe, T.X., Nakajima, K., Sakakibara, S., Takao, T. and Shimonishi, Y. 1990a. Amino acid sequence and relative biological activity of a natriuretic peptide isolated from eel brain. Biochem. Biophys. Res. Commun. 170: 883-891.
Westenfelder, C., Birch, F.M., Baranowski, R.L., Rosenfeld, M.J., Shiozawa, D.K. and Kablitz, C. 1988. Atrial natriuretic factor and salt adaptation in the teleost fish Gala atraria. Am. J. Physiol. 255: F1281-1286.
