Teleost-type angiotensin is present in Australian lungfish, Neoceratodus forsteri.

Angiotensin I (ANG I) was produced from the incubation of lungfish plasma with homologous kidney extracts. The purified peptide was found to have the sequence of H-Asn-Arg-Val-Tyr-Val-His-Pro-Phe-Thr-Leu-OH, which is homologous for the first eight residues with all teleost angiotensins so far sequenced, although lungfish generally possess tetrapod-type hormones. The lungfish decapeptide (ANG I) induced dose-dependent increases in arterial pressure in the rat. The lungfish octapeptide (ANG II) released aldosterone from kidney-adrenal tissue in vitro in a dose-dependent manner and induced dose-dependent increases in arterial pressure of the lungfish. Substitution of asparagine with aspartic acid in the first position (tetrapod-type ANG II) did not alter the blood pressure response significantly, but a second substitution of the valine in the (5)-position with isoleucine (ANG II form found in human and rat) abolished the rise in arterial pressure in lungfish over the same dose range.

Tetrapod-type [Asp1] angiotensin is present in a holostean fish, Amia calva.

The renin-angiotensin system has been identified in various vertebrates, from elasmobranchs to mammals. Tetrapod (amphibians to mammals) angiotensin (ANG) has Asp at the N-terminus, but Asp is replaced by Asn in elasmobranch and teleost fish. ANG I has been isolated from incubates of plasma and kidney extracts of the bowfin Amia calva, a holostean fish, using the eel vasopressor activity as an assay system; its sequence was found to be H-Asp-Arg-Val-Tyr-Val-His-Pro-Phe-Asn-Leu-OH after sequence analysis, mass spectrometry, and comparison with the synthetic peptide. This sequence is identical to bullfrog ANG I. [Asn1] ANG I was not detected. Thus the bowfin is the first fish species which contains only [Asp1] ANG I. The bowfin ANG I and II were no more vasopressor than eel peptides in the bowfin, indicating that bowfin ANG II receptors do not distinguish between [Asp1] and [Asn1] peptides. In the rat, bowfin ANG I and rat [Ile5, His9] ANG I have equipressor activities when examined in different animals, but the vasopressor activity of bowfin ANG I decreased following rat ANG I in the same animals, although the activity of rat ANG I was unaffected after bowfin ANG I. The present study directly demonstrates the presence of the renin-angiotensin system in a holostean fish and showed that its ANG II receptors have not yet fully coevolved with the homologous [Asp1] peptide.

A new natriuretic peptide isolated from cardiac atria of trout, Oncorhynchus mykiss.

Atrial and brain natriuretic peptides (ANP and BNP, respectively) are two cardiac natriuretic peptides (NPs) found in tetrapods from amphibians to mammals, whereas ANP and ventricular NP (VNP) have been identified in eel hearts. Because VNP has also been found in the rainbow trout ventricle, we attempted to isolate NP from trout cardiac atria in order to determine whether ANP and VNP are common cardiac NPs in teleosts. In the present experiments, we isolated VNP and a novel atrial NP consisting of 29 amino acid residues from the atria. This new trout NP exhibited similar sequence identity to mammalian ANP and BNP (50-60%). Its homology to eel ANP was low (52%) compared with high homology of trout and eel VNP (78%). Based on yield, the content of this new NP in trout atria may be even smaller than that of VNP. The new trout atrial NP exhibited low relaxant activity in the chick rectum (only 1/10 of that of trout VNP), and extremely low vasorelaxant activity in the rat aortic strip (only 1/400 of that of human ANP). However, the new trout NP was equipotent with trout VNP and human ANP in relaxing trout epibranchial artery. Based on the sequence similarity with other NPs and on atrial content, the new NP isolated from trout atria cannot yet be assigned to a known member of the NP family.

B-type natriuretic peptide isolated from frog cardiac ventricles.

A new natriuretic peptide with 27 amino acid residues has been isolated from cardiac ventricles of the bullfrog, Rana catesbeiana. Since this ventricular peptide had high sequence identity to B-type (brain) natriuretic peptide (BNP), especially to chicken BNP (74%), we named it bullfrog BNP. Thus, semi-aquatic amphibians have tetrapod-type BNP, but do not seem to have fish-type ventricular natriuretic peptide (VNP) in their ventricles. Compared with other known BNPs, the C-terminus of bullfrog BNP was elongated by two amino acid residues and was not amidated. Bullfrog BNP dose-dependently decreased arterial blood pressure in the bullfrog with a potency twofold greater than that of human ANP. Bullfrog BNP also exhibited vasodepressor, natriuretic and diuretic activities in the rat, but it was 1/3, 1/7, and 1/17 as potent as human ANP in this mammalian species.

Vasopressor activities of N-terminal fragments of adrenomedullin in anesthetized rat.

Adrenomedullin (AM) is a vasorelaxant peptide that was recently isolated from human pheochromocytoma. In contrast to human (h) AM, which has vasodepressor activity, a synthetic N-terminal fragment of hAM, hAM-(1-25)-NH2 showed vasopressor activity in the anesthetized rat. The N-terminal peptides hAM-1-31)-NH2, hAM-(1-25)-OH, hAM-(1-21)-NH2, acetyl-hAM-(16-21)-NH2, and acetyl-hAM-(16-36)-OH all showed vasopressor activities. The potency of hAM-(1-21)-NH2, acetyl-hAM-(16-21)-NH2 was greater than that of hAM-(1-25)-NH2. Pretreatment with phenoxybenzamine, guanethidine, or reserpine attenuated vasopressor activities of these peptides. These data suggested that vasopressor activity of N-terminal fragment of hAM is due to a stimulation of endogenous catecholamine release.

Smooth muscle relaxing and hypotensive activities of synthetic calciseptine and the homologous snake venom peptide FS2.

The biological activities of synthetic calciseptine and FS2, a homologous peptide from snake venom, were determined using in vitro and in vivo preparations. Calciseptine and FS2 produced dose-dependent relaxation in pre-constricted rat aorta, pulmonary artery and trachea. The onset and duration pattern of these relaxing effects were similar to those caused by nifedipine, an L-type Ca2+ channel blocker. Calciseptine relaxed the contraction of rat aorta provoked by an L-type channel agonist, Bay K 8644. This relaxation was not affected by NG-nitro-L-arginine, indomethacin or propranolol. Calciseptine and FS2 inhibited the contraction caused by acetylcholine in guinea pig ileal longitudinal muscle. In case of in vivo study using anesthetized rats, calciseptine, FS2 and nifedipine showed depressor effects. The hypotensive effects of the two peptides were more potent and sustained than that of nifedipine. These findings show that both synthetic calciseptine and FS2 have similar biological activities like nifedipine, an L-type Ca2+ channel blocker. In addition, these two peptides with large molecular weights may be unique and useful tools for studying the Ca2+ channel.

Rainbow trout ventricular natriuretic peptide: isolation, sequencing, and determination of biological activity.

Ventricular natriuretic peptide (VNP) is a new type of cardiac natriuretic peptide initially isolated from the eel ventricle. VNP has been isolated from cardiac ventricles of the rainbow trout, Oncorhynchus mykiss, and found to consist of 35 amino acid residues carrying a C-terminal tail sequence with 14 amino acid residues. Thus, the long C-terminal sequence characteristic of VNP was also conserved in the trout VNP. A VNP with 4 amino acid residues truncated from the C-terminus was also isolated from trout ventricles and sequenced. Sequence identity of trout VNP to eel VNP was 77%; while it was 56% to eel A-type natriuretic peptide (ANP). Trout VNP caused characteristic biphasic vasopressor/depressor effects in the trout similar to those produced by rat and eel ANP. Trout VNP and human ANP were almost equipotent in their vasopressor and depressor activity in trout. Unlike eel VNP, therefore, homologous VNP did not exhibit greater activity in the trout. In the rat, however, trout VNP was more potent than eel peptide and was almost equipotent to human ANP for both vasodepressor and natriuretic effects. The high potency of trout VNP appears to be due in part to its longer-lasting effect compared to human ANP.

The biological activity of endothelin-1 analogues in three different assay systems.

Endothelin (ET) is a vasoconstrictor peptide with 21-amino acid residues. In this study, we determined the relative potencies of ET-1 analogues to investigate the essential moiety of ET-1 for expression of its biological effect. We synthesized ET-1 analogues with the substitution of one amino acid at positions 2-18 and 21. All ET-1 analogues had the proper intramolecular disulfide bonds. We have conducted a systematic survey to compare the biological activity of ET-1 analogues in three different assay systems: the vasoconstrictor activity in the rat pulmonary artery, the nonvascular smooth muscle contracting activity in the rat trachea, and the pressor activity in the conscious rat. In the pulmonary arterial preparation, the carboxyl groups of Asp8, Glu10, and Asp18, the aromatic groups of Phe14, His16, and Trp21, and the hydrophobic group of Leu17 contributed to the expression of the vasoconstrictor activities. However, the contracting activities in the tracheal preparations were retained even upon replacement of these amino acid residues by alanine or other amino acids. Moreover, substitution of the amino acid residue of ET-1 at positions Ser5, Lys9, and Tyr13 for alanine was found to increase the potency of ET-1 by a factor of about 2. With respect to vasopressor effects, the activities were retained, and all ET-1 analogues showed relative potencies ranging from 5 to 90% of ET-1. These findings suggest that different types of receptors may be present in different organs.