Biomedical subjects

T X Watanabe

Publications and source records attributed to T X Watanabe.

At least 37 records · Page 2Linked to original sources

PubMed1993

Goldfish calcitonin: purification, characterization, and hypocalcemic potency.

A novel teleost calcitonin was isolated from the goldfish, Carassius auratus, and its amino acid sequence was determined to be H-Cys-Ser-Ser-Leu-Ser-Thr-Cys-Val-Leu-Gly- Lys-Leu-Ser-Gln-Glu-Leu-His-Lys-Leu-Gln-Thr-Tyr-Pro-Arg-Thr-Asn-Val-Gly- Ala-Gly-Thr-Pro-NH2. The structure of goldfish calcitonin (gCT) was similar to that of salmon calcitonin (sCT) but differed from the latter at positions 3, 27, and 29. The hypocalcemic activity of gCT was estimated to be 3,470 IU/mg by the standardized rat bioassay method, compared with a value of 3,500 IU/mg for sCT. After administration of gCT to young goldfish, the serum Ca concentration was significantly lower in some cases than in controls. However, in other cases no such difference was recognized.

Amino Acid Sequence↗

PubMed1993

New angiotensin I isolated from a reptile, Alligator mississippiensis.

Angiotensin I (ANG I) of the American alligator Alligator mississippiensis was isolated from incubates of homologous plasma and kidney extract, and its amino acid sequence was determined as H-Asp-Arg-Val-Tyr-Val-His-Pro-Phe-Ala-Leu-OH. The presence of strongly hydrophobic alanine at position 9 is unusual among ANGs I sequenced to date. Since alanine can be converted to serine by a one-point mutation of the triplet nucleotides, the phylogenetic proximity of the alligator to birds, whose ANG I has serine at position 9, is confirmed by the chemical evolution of the hormone structure. The alligator [Asp1,Val5,Ala9] ANG I increased arterial blood pressure of conscious alligators, but it was no more potent than avian [Asp1,Val5,Ser9] ANG I. The alligator ANG I was also equipotent to the homologous ANG I in the quail and rat. It is concluded that substitutions at position 9 have little influence on the vasopressor activity of ANG I.

Alligators and Crocodiles↗

PubMed1993

Central cardiovascular effects of joining peptide in genetically hypertensive rats.

Joining peptide (JP) is one of the major products of proopiomelanocortin (POMC). The biological function of this peptide has not been clarified despite its relative abundance in the pituitary and the hypothalamus. Recently we demonstrated that JP, which was isolated from bovine posterior pituitary, possesses Na pump inhibitor activity. The purpose of this study is to explore the physiological relevance of JP in cardiovascular regulation. For these investigations, we used the synthetic peptides bovine JP (bJP) and COOH-terminally amidated rat JP (rJP), since JP is known to have sequence variability among species. Intracisternal administration of both bJP and rJP in urethan-anesthetized rats evoked similar hypertensive and tachycardia effects. The effects of both peptides were markedly greater in the spontaneously hypertensive rats (SHR) compared with the normotensive Wistar Kyoto rats (WKY). Intravenous bolus injections of rJP at the same doses were without effect. Autoradiography, using 125I-labeled [0Tyr]-rJP as a ligand, revealed specific binding sites for rJP in the dorsal medulla in areas corresponding to the nucleus tractus solitarii (NTS) (extending from approximately 0.4 mm caudal to 1.8 mm rostral to the obex). Microinjections of rJP into the caudal part of the NTS of anesthetized SHR produced dose-related pressor and tachycardic responses. The pressor and tachycardic responses were also observed at the rostral part of the NTS, whereas the injections into the intermediate part of the NTS evoked depressor and bradycardic responses in SHR. These results suggest that at doses tested, the site of JP action resides in the central nervous system, and that JP is a potent neuropeptide in medullary sites known to be pivotal in central cardiovascular regulation. The effect of JP is especially prominent in the genetically hypertensive rat.

Analysis of Variance↗

PubMed1993

A novel angiotensin I isolated from an elasmobranch fish.

It is believed that the renin-angiotensin system evolved initially in primitive bony fishes and is absent from elasmobranchs. We have isolated angiotensin I from the incubates of plasma and kidney extracts of an elasmobranch fish, Triakis scyllia, using eel vasopressor activity as an assay system. Its sequence was determined to be H-Asn-Arg-Pro-Tyr-Ile-His-Pro-Phe-Gln-Leu-OH. Dogfish angiotensin I is teleost-like because of an asparagine residue at position 1 but it is mammalian-like because of an isoleucine residue at position 5. The unique and most important substitution in dogfish angiotensin I is a proline residue at position 3 which may cause significant changes in its tertiary structure. A glutamine residue at position 9 is also unique among all angiotensin Is sequenced to date. Dogfish angiotensin I is more potent than rat angiotensin I in its vasopressor activity in the dogfish but the relationship is reversed in the rat. Thus angiotensin receptors as well as the hormone molecules appear to have evolved during vertebrate phylogeny. Our findings establish the elasmobranch renin-angiotensin system and support the hypothesis that the renin-angiotensin system is a phylogenetically old hormonal system which plays important roles in cardiovascular and fluid homeostasis.

Amino Acid Sequence↗

PubMed1992

Calcitonin of the stingray: comparison of the hypocalcemic activity with other calcitonins.

Hypocalcemic potency of calcitonin isolated from the stingray (cartilaginous fish), Dasyatis akajei, was examined using the rat bioassay and compared with the activities of other calcitonins (human, pig, salmon, eel, and fowl). The potency of Dasyatis calcitonin (dCT) was estimated to be 1500-3800 IU/mg. However, when the duration of the hypocalcemic effect of dCT was taken into consideration, it was judged that dCT was approximately 2.4-6.2 times more potent than mammalian calcitonins, but about 2.3-3.5 times less active than other nonmammalian calcitonins.

Amino Acid Sequence↗

PubMed1992

Ca2+ release from isolated sarcoplasmic reticulum of guinea-pig psoas muscle induced by K(+)-channel blockers.

A Ca(2+)-sensitive electrode was used to measure the Ca2+ concentration of the medium containing the heavy fraction of the fragmented sarcoplasmic reticulum (SR) prepared from guinea-pig psoas muscle. Among K(+)-channel blockers tested, 4-aminopyridine (4-AP), tetraethylammonium (TEA) and charybdotoxin elicited Ca2+ release from the SR, but apamin and glibenclamide did not. These results suggest that a reduction of SR K+ conductance leads to Ca2+ release from the SR.

4-Aminopyridine↗

PubMed1992

A C type natriuretic peptide is a vasodilator in vivo and in vitro in the common dogfish.

The effects of an elasmbranch cardiac C-type natriuretic peptide (dogfish CNP-22) on arterial blood pressure were investigated in vivo in chronically cannulated dogfish Scyliorhinus canicula and in vitro by a myographic technique using the distal part of the first branchial artery. In-vivo dogfish CNP-22 caused a dose-dependent reduction in mean arterial blood pressure which was much more potent than that of alpha-human ANP. In-vitro dogfish CNP-22 also caused a dose-dependent relaxation which was independent of the endothelium. These results are in marked contrast to those obtained in similar studies on other vertebrate species in which CNP exhibited only mild hypotensive effects compared to both atrial and brain natriuretic peptides. This study indicates the importance of using homologous peptides in determining the physiological role of natriuretic peptides in non-mammalian vertebrates.

Animals↗

PubMed1991

A novel natriuretic peptide isolated from eel cardiac ventricles.

A new natriuretic peptide, which exhibits the entire spectrum of actions known to be characteristic of atrial and brain natriuretic peptides (ANP and BNP), was isolated from eel cardiac ventricles and has been named ventricular natriuretic peptide (VNP). The primary structure of eel VNP is characterized by its uniquely long C-terminal 'tail' that extends from the second half-cystine. Thus, eel VNP appears to be a novel natriuretic peptide of a type not found in mammals. With respect to natriuretic (rat) and vasodepressor (rat and eel) activities, eel VNP is much more potent than human ANP in eels and almost equipotent in rats. Strong tachyphylaxis is observed for the vasodepressor effect in both rats and eels, whereas it is not observed for the natriuretic effect in rats.

Amino Acid Sequence↗

PubMed1991

The cystine-stabilized alpha-helix: a common structural motif of ion-channel blocking neurotoxic peptides.

Neurotoxic peptides from venoms of scorpions and honey bees exhibit a consensus pattern in the two disulfide bridgings related to the sequence portions Cys-X-Cys and Cys-X-X-X-Cys. A revised three-dimensional structure of charybdotoxin, as determined by two-dimensional nmr spectroscopy, confirms that the consensus cystine dislocation generates in all these toxins a common structural element, i.e., the cystine-stabilized alpha-helical (CSH) motif, which may be correlated with their common ion channel blocking activity.

Amino Acid Sequence↗

PubMed1991

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.

Amino Acid Sequence↗

PubMed1990

Solution synthesis of charybdotoxin (ChTX), a K+ channel blocker.

Charybdotoxin, a 37 amino acid peptide which is a minor component of Leiurus quinquestriatus venom, was synthesized by the solution procedure applying our maximum protection strategy. After formation of the three disulfide bonds, for which a redox buffer was necessary, the final product was purified to homogeneity and found to have similar biological potency to that reported by others for the natural product. The disulfide bond configuration was found to be: Cys7-Cys28; Cys13-Cys33; Cys17-Cys35. Conformational analysis by 1H-NMR showed that the molecule exists as a very tightly folded structure, in which residues 1-7 and 24-37 form a triple-stranded beta-sheet, with a turn at positions 30-31. The region from 11-20 appears to adopt an alpha-helical conformation.

Amino Acid Sequence↗

PubMed1990

Amino acid sequence and relative biological activity of a natriuretic peptide isolated from eel brain.

A peptide exhibiting natriuretic and vasodepressor activity was isolated from eel brains. Its amino acid sequence was found to be similar to, but distinct from, that of the eel atrial natriuretic peptide (ANP), and it is characterized by the absence of the C-terminal sequence that follows the second-half cystine. The extent of sequence homology of this peptide to known mammalian brain natriuretic peptides (BNPs) is greater than to ANPs. Therefore, we have named this peptide "eel BNP-like peptide". The isolation of this peptide provides the first evidence for the presence of two different molecular types of natriuretic peptide in a single non-mammalian species. The potency of the eel BNP-like peptide relative to that of human ANP, in terms of its vasodepressor activity, was 117 in the eel, 1.7 in the quail, and 0.08 in the rat. Thus, eels exhibit a high degree of sensitivity to this native peptide just as they do to eel ANP.

Amino Acid Sequence↗

PubMed1990

Receptor binding and vasoconstrictor activity of big endothelin.

We studied the effects of synthetic porcine big endothelin (ET), a putative 39-residue intermediate deduced from cDNA sequence analysis, on receptor binding activity in cultured rat vascular smooth muscle cells as well as its vasoconstrictive activity in rat pulmonary artery. Big ET was about two orders of magnitude less active than ET in the receptor binding and vasoconstriction assays. Our data are consistent with the contention that big ET is an intermediate which can be converted into fully bioactive 21-residue mature ET.

Animals↗

PubMed1989

Amino acid sequence and relative biological activity of eel atrial natriuretic peptide.

A peptide exhibiting vasodepressor and natriuretic activities in rats was isolated from eel atria, and its primary structure was determined as H-Ser-Lys-Ser-Ser-Ser-Pro-Cys-Phe-Gly-Gly-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Tyr-Ser- Gly-Leu-Gly-Cys-Asn-Ser-Arg-Lys-OH. This peptide, termed eel atrial natriuretic peptide (ANP), has sequence homology of 59% to mammalian (human or rat) ANP, 52% to fowl ANP, and 46% to frog ANP. When the biological activity of synthetic eel ANP was compared with that of human ANP, the eel peptide was 110 times more potent for the vasodepressor activity in eels, nearly equipotent for the vasodepressor activity in quails, and 20 times less potent for the vasodepressor and natriuretic activity in rats.

Amino Acid Sequence↗

PubMed1989

Structure-activity relationship of endothelin: importance of charged groups.

Endothelin (ET)-related peptides including ET-1 (1-39) were synthesized, and their constricting activity in rat pulmonary artery rings and pressor activity in unanesthetized rat were measured to elucidate their structure-activity relationship. The vasoconstrictor activities of ET-2, ET-3 and sarafotoxin S6b were one-half, one-60th and one-third that of ET-1, respectively. Such differences in biological activities should mainly arise from sequence heterogeneity at the N-terminal portion, especially at positions 4 to 7. All of the blocked ETs at the amino or carboxyl termini showed greatly decreased activities. A monocyclic analog, in which Cys3 and Cys11 were replaced by Ala, showed one-third the activity of ET-1; however, its deamino dicarba analog was almost completely inactive. Significant activities were retained even with replacement of amino acids at positions Ser4, Ser5, Leu6, Met7, Lys9, Tyr13, and Trp21 by Ala, Ala, Gly, Met(0), Leu, Phe, and Tyr or Phe, respectively. On the other hand, replacement of Asp8, Glu10 and Phe14 by Asn, Gln and Ala, respectively, resulted in complete loss of the biological activity. These results indicated that two disulfide bonds in ET molecule were not essential for the expression of vasoconstricting activity. Both terminal amino and carboxyl groups, carboxyl groups of Asp8 and Glu10, and the aromatic group of Phe14 seemed to be contributing, more or less, to the expression of the biological activities.

Animals↗

PubMed1989

Interaction of synthetic sarafotoxin with rat vascular endothelin receptors.

The effects of synthetic analogs of sarafotoxin (STX) S6b, a snake venom peptide with a high sequence homology to the endothelium-derived vasoconstrictor endothelin (ET), on ET receptor binding activity and cytosolic free Ca2+ concentration [( Ca2+]i) were studied in cultured rat vascular smooth muscle cells. Binding studies revealed that [Cys1-15, Cys3-11] STX competed with 125I-ET for the binding to its vascular receptors with lower affinity than that of ET, but was far more effective than [Cys1-11, Cys3-15]STX in inhibiting the binding. [Cys1-15, Cys3-11]STX had a less potent effect on increasing [Ca2+]i than ET, whereas [Cys1-11, Cys3-15]STX was inactive. These data suggest that there may exist heterogenous subpopulations of the vascular ET/STX receptors, and that the proper double cyclic structure of STX is essential for interacting with its putative receptors to induce the [Ca2+]i response.

Animals↗

PubMed1989

Receptor binding activity and cytosolic free calcium response by synthetic endothelin analogs in cultured rat vascular smooth muscle cells.

Using a variety of synthetic analogs of porcine endothelin (pET), we have studied the effects of these analogs on receptor binding activity and cytosolic free Ca2+ concentrations ([Ca2+]i) in cultured rat vascular smooth muscle cells (VSMC). Removal of C-terminal Trp21 residue, truncated derivatives pET(1-15) and (16-21), substitution of disulfide bond, Cys(3-11) or Cys(1-15), by Cys (Acm), all resulted in a complete loss of receptor binding activity and [Ca2+]i response, while N-terminal elongation of Lys-Arg residues, but not oxidation of Met7 residue, decreased receptor binding activity and [Ca2+]i response. [Cys1-15,Cys3-11]pET was far more potent than [Cys1-11,Cys3-15]pET in receptor binding and [Ca2+]i response. These data indicate that the C-terminal Trp21 as well as the proper double cyclic structure formed by the intramolecular disulfide bonds of the pET molecule are essential for receptor binding and subsequent [Ca2+]i increase in rat VSMC.

Animals↗

PubMed1989

Synthesis of endothelin-1 analogues, endothelin-3, and sarafotoxin S6b: structure-activity relationships.

Two disulfide analogues (types A and B) of endothelin-3 (ET-3; formerly, rat ET), sarafotoxin S6b, and apamin, were synthesized to determine their disulfide structures as in the case of endothelin-1 (ET-1; formerly human and porcine ET). The disulfide structures of ET-3 and sarafotoxin S6b were found to be identical with that of ET-1 (type A) but distinct from that of apamin (type B). The vasoconstricting activities of ET-3 and sarafotoxin S6b were about one-60th and one-third that of ET-1, respectively. Such different biological potencies between endothelins and sarafotoxin S6b could be largely attributed to the sequence heterogeneity at the N-terminal portion. ET-1 analogues were also synthesized to clarify the structure-activity relationships. The opening of any disulfide bond in the ET-1 molecule extremely decreased the activity, while oxidation of the Met residue did not alter it. Amidation of the terminal COOH group and extension of the Lys-Arg sequence to the N-terminus led to 16- and 540-fold decreases in activity, respectively. Removal of the C-terminal Trp residue resulted in complete loss of the activity. The other disulfide analogues (type B and C) of ET-1 showed markedly lower activity than the parent molecule (type A). These results indicated the importance of the whole molecule with the proper double cyclic structure for determining its active conformation.

Amino Acid Sequence↗