Glucagon-like peptide isolated from the eel intestine: effects on atrial beating
1 Laboratory of Integrative Physiology, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan,
2 Tokyo Research Laboratories, Kyowa Hakko Kogyo Co. Ltd, 3-6-6 Asahimachi, Machidashi, Tokyo 194-0023, Japan and
3 Department of Environment and Mutation, Research Institute for Radiation Biology and Medicine, Hiroshima University, Hiroshima 734-8553, Japan
*Author for correspondence (e-mail: mando{at}hiroshima-u.ac.jp)
Accepted June 13, 2001
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Summary |
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Key words: eel, oxyntomodulin, intestine, atrial beating, intracellular Ca2+, extracellular Ca2+, Anguilla japonica.
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Introduction |
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We have determined that the eel atrium provides a suitable bioassay system, because it is both easy to prepare and is sensitive to peptides. The eel atrium can be easily isolated from the heart and beats spontaneously for more than 10h in artificial saline. In addition, it has been demonstrated that the isolated eel atrium is sensitive not only to adrenaline or acetylcholine (ACh) (Yasuda et al., 1996), but also to eel neuropeptide Y (eNPY) (Uesaka, 1996). As a candidate intestinal hormone capable of enhancing atrial beating, we have isolated a glucagon-like peptide (HSQGTFTNDY10SKYLETRRAQ20DFVQWLMNSK30RSGGPT) from the eel intestine. This peptide is shown to exhibit positive inotropic and chronotropic actions in the atrium. The inotropism is due to an increase in intracellular free Ca2+ concentration ([Ca2+]i).
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Materials and methods |
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Bioactive fractions were pooled and subjected to high-performance liquid chromatography (HPLC) separation (LC-6AD, Shimadzu, Kyoto, Japan). Retained material was eluted with a 50min linear gradient of 0%90% acetonitrile containing 10% 2-propanol and 0.1% TFA, and each fraction was bioassayed. The active fractions were applied to a C18 reverse-phase column (TSK ODS-80TM, Tosoh) and eluted with a 100min linear gradient of 15%35% acetonitrile containing 5% 2-propanol and 0.1% TFA. The active fractions were further applied to a cation-exchange column (TSK CM-5PW, Tosoh) and eluted with a 35min linear gradient of 00.35moll-1 NaCl in 10% 2-propanol and 20mmoll-1 phosphate buffer (pH 6.8). The bioactive peak was rechromatographed on the C18 reverse-phase column (TSK ODS-80TM) with a 50min linear gradient of 24%34% acetonitrile containing 5% 2-propanol and 0.1% TFA. Final purification was performed using the same column under isocratic conditions, as shown in Fig.1A, to give a single peak, EI-14.
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Measurement of biological activity in the eel atrium
Japanese eels, weighing approximately 220g, were kept in sea water (20°C) for more than 1 week. After decapitation, the heart was rapidly excised and the atrium was isolated on ice. It was then tied with two cotton threads and connected to a force transducer (type 451996, Sanei, Tokyo, Japan). The details of this procedure have been described previously (Uesaka, 1996; Yasuda et al., 1996). The isolated eel atrium was bathed in artificial saline consisting of (in mmoll-1): 118.5 NaCl, 4.7 KCl, 3.0 CaCl2, 1.2 MgCl2, 1.2 KH2PO4, 24.9 NaHCO3 and 10 sodium lactate, bubbled with a 95% O2/5% CO2 gas mixture (pH 7.4) at room temperature (2428°C). Although the pH of fish blood is approximately pH 7.9, equilibrated with 0.5% CO2 (Farrell, 1984), experiments in the present study were usually performed in the presence of 5% CO2 for convenience, since we have established that the effects of adrenaline, ACh, eNPY and eel oxyntomodulin (eOXM) were similar in the presence of 5% CO2 (pH 7.4) and 0.5% CO2 (pH 7.9) (T. Uesaka and M. Ando, unpublished observation). In Ca2+-free Ringers solution, 3.0mmoll-1 CaCl2 was replaced with 4.5mmoll-1 NaCl. After preloading by 5.9mN, spontaneous isometric contractions were converted into electrical signals by a transducer connected to a strain amplifier (6M82, Sanei) and these were recorded using an electric polyrecorder (EPR-10B, Toa, Tokyo, Japan). The rate of contraction was measured simultaneously using a tachometer (type 1321, Sanei). When examining atrial contractions in the absence of Ca2+, the preparation was flushed twice with a Ca2+-free Ringers solution.
The effects of adrenaline (Sigma Chemical, St Louis, MO, USA), betaxolol (Mitsubishi Kasei, Tokyo, Japan) and verapamil HCl (Wako Pure Chemical, Osaka, Japan) were also examined.
Measurement of intracellular free Ca2+ concentration
[Ca2+]i in the myocardium was measured as described previously (Uesaka, 1996). Briefly, the isolated atrium was cut into pieces in Ringers solution and treated with collagenase (1mgml-1; Wako Pure Chemical) in the same solution for 1h at room temperature. After rinsing with Ringers solution, the partially dispersed tissue was incubated with Calcium Green-1 acetoxymethyl ester (5µmoll-1; Molecular Probes, Eugene, OR, USA) and with Pluronic F-127 (0.05%, Molecular Probes) as a surfactant for 40min. The atrial cells were stuck onto a glass slide using coverslips, and mounted in a small chamber on the stage of an inverted microscope (Axiovert 135 MTV, Zeiss, Tokyo, Japan) and superfused with normal Ringers solution. The fluorescent signal from Calcium Green-1 was recorded with a laser scanning confocal imaging system (MRC-600, Bio-Rad, Tokyo, Japan; dichroic reflecter 510LP; emission filter 515LP) equipped with a KrAr laser (5470K, Ion Laser Technology, Salt Lake City, UT, USA; 488nm for excitation). The intensity of the fluorescence was expressed in arbitrary units ranging from 0 to 255.
Statistical analyses
Data are reported as means ± S.E.M. N represents the number of preparations. The statistical significance of difference between means was examined using a MannWhitney U-test. The null hypothesis was rejected for P<0.05.
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Results |
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Table2 shows the amino acid composition of EI-14, which was almost identical to that expected from the sequence analysis described above. The relative molecular mass (Mr) of EI-14 (4207±1) was almost identical to that predicted from the sequence (Mr=4207). To identify the structure, a peptide consisting of the 36 amino acid residues was synthesized using the sequence result. Fig.2 compares the synthesized peptide (S) with native EI-14 (N) on the same HPLC. The retention time of the synthesized peptide was identical to that of the native EI-14 in both reverse-phase and cation-exchange HPLC. When a mixture of native and synthesized peptide was applied, only a single peak was observed with both types of HPLC.
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Discussion |
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The eel glucagon-like peptide (eOXM) has positive inotropic and chronotropic effects on the eel atrium (Fig.1B, Fig.5A). Although these effects of eOXM are similar to those of adrenaline (Yasuda et al., 1996), eOXM appears to stimulate atrial beating independently of the ß1-adrenoceptors. In the presence of betaxolol, a ß1-adrenoceptor antagonist, the effects of eOXM remain (present study), while the effects of adrenaline are completely blocked, as previously described (Uesaka, 1996).
Eel oxyntomodulin increases [Ca2+]i in the eel myocardium (Fig.4). Similar increases in [Ca2+]i are observed after treatment of the eel atrium with adrenaline or neuropeptide Y (Uesaka, 1996). It is thus plausible that eOXM enhances contractility via an increase in [Ca2+]i. In the eel atrium, contraction appears to be induced by entry of Ca2+ from the extracellular fluid, since atrial contractility is markedly reduced in Ca2+-free Ringers solution or after treatment with verapamil, a Ca2+ channel blocker (Fig.5). In general, heart contractions in poikilotherms can be induced by an influx of Ca2+ from the extracellular fluid. Electron microscopy of a number of poikilotherm hearts demonstrates both a sparsity of sarcoplasmic reticulum and an absence of transverse tubules (Santer, 1985). Furthermore, contraction of the amphibian (Bers, 1985) and teleost (Driedzic and Gesser, 1988) ventricle appears to be relatively insensitive to ryanodine, a blocker of the sarcoplasmic reticulum Ca2+ release. More directly, Mery et al. (Mery et al., 1990) demonstrated, using a patch-clamp technique, that the Ca2+ current is increased by glucagon in frog and rat ventricular myocytes. It is likely therefore, that eOXM enhances Ca2+ influx, enhancing [Ca2+]i, which enhances the contractile force of the eel atrium. However, the chronotropic effect of eOXM is still present after Ca2+ entry has been inhibited with Ca2+-free solutions or verapamil. This indicates that the chronotropic effect of eOXM is independent of extracellular Ca2+. Eel oxyntomodulin might stimulate beating rate by increasing levels of other intracellular mediators, such as cyclic AMP. The fact that the oscillations in [Ca2+]i are much slower than the beating rate in our preparations may be because of damage to the pacemaker cells by collagenase and/or Pluronic F-127.
Eel oxyntomodulin may act as a hormone, being produced in the intestine and targeted to the heart. It is not known what triggers release of eOXM from the intestine, but a recent finding in the rat small intestine is of value for reference. When the lumen of the small intestine is perfused with 200mmoll-1 NaCl Ringers solution (hyperosmotic), guanylin (an intestinal peptide composed of 15 amino acid residues) secretion into the lumen increased threefold, accompanied by a slight increase in uroguanylin secretion (Kita et al., 1999). Uroguanylin is another intestinal peptide of 15 amino acid residues and is considered to be a hormone targeting the kidney and pancreas (Nakazato et al., 1998), while guanylin is a luminocrine substance (Forte and Currie, 1995). If similar factors, such as concentrated NaCl, stimulate eOXM secretion from the intestine into the circulation, eOXM may enhance the contractile force and beating rate of the heart.
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Acknowledgments |
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References |
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Abad, M. E., Peeze Binkhorst, F. M., Elbal, M. T. and Rombout, J. H. W. M. (1987). A comparative immunocytochemical study of the gastro-entero-pancreatic (GEP) endocrine system in a stomachless and a stomach-containing teleost. Gen. Comp. Endocrinol. 66, 123136.[Medline]
Barragan, J. M., Rodriguez, R. E. and Blazquez, E. (1994). Changes in arterial blood pressure and heart rate induced by glucagon-like peptide-1-(7-36) amide in rats. Am. J. Physiol. 266, E459E466.
Bataille, D., Gespach, C., Tatemoto, K., Marie, J. C., Caudray, A. M., Rosselin, G. and Mutt, V. (1981). Bioactive enteroglucagon (oxynto-modulin): present knowledge on its chemical structure and its biological activities. Peptides 2, 4144.[Medline]
Bataille, D., Tatemoto, K., Gespach, C., Jonvall, H., Rosselin, G. and Mutt, V. (1982). Isolation of glucagon-37 (bioactive enteroglucagon/oxyntomodulin) from porcine jejuno-ileum. Characterization of the peptide. FEBS Lett. 146, 7986.[Medline]
Bers, D. M. (1985). Ca influx and sarcoplasmic reticulum Ca release in cardiac muscle activation during postrest recovery. Am. J. Physiol. 248, H366H381.[Medline]
Chernow, B., Reed, L., Geelhoed, G. W., Anderson, M., Teich, S., Meyerhoff, J., Beardsley, D., Lake, C. R. and Holaday, J. W. (1986). Glucagon: endocrine effects and calcium involvement in cardiovascular actions in dogs. Circulation Shock 19, 393407.
Conlon, J. M., Dafgard, E., Falkmer, E. and Thim, L. (1987). A glucagon-like peptide, structurally related to mammalian oxyntomodulin, from the pancreas of a holocephalan fish, Hydrolagus colliei. Biochem. J. 245, 851855.[Medline]
Conlon, J. M., Deacon, C. F., Hazon, N., Henderson, I. W. and Thim, L. (1988). Somatostatin-related and glucagon-related peptides with unusual structural features from the European eel (Anguilla anguilla). Gen. Comp. Endocrinol. 72, 181189.[Medline]
Conlon, J. M., Hazon, N. and Thim, L. (1994). Primary structures of peptides derived from proglucagon isolated from the elasmobranch fish, Scyliorhinus canicula. Peptides 15, 163167.[Medline]
Driedzic, W. R. and Gesser H. (1988). Differences in forcefrequency relationships and calcium dependency between elasmobranch and teleost hearts. J. Exp. Biol. 140, 227241.
Elbal, M. T. and Agulleiro, B. (1986). An immunocytochemical and ultra- structural study of endocrine cells in the gut of a teleost fish, Sparus auratus L. Gen. Comp. Endocrinol. 64, 339354.[Medline]
Farah, A. E. (1983). Glucagon and heart. In Handbook of Experimental Pharmacology, vol. II (ed. P. Lefevre), pp. 553609. Berlin: Springer-Verlag.
Farah, A. E. and Tuttle, R. (1960). Studies of the pharmacology of glucagon. J. Pharmacol. Exp. Thr. 129, 4955.
Farrell, A. P. (1984). A review of cardiac performance in the teleost heart: intrinsic and humoral regulation. Can. J. Zool. 62, 523536.
Forte, L. R. and Currie, M. G. (1995). Glucagon: a peptide regulator of epithelial transport. FASEB J. 9, 643650.
Iwanij, V. and Hur, K. C. (1987). Development of physiological responsiveness to glucagon during embryogenesis of avian heart. Dev. Biol. 122, 146152.[Medline]
Kita, T., Kitamura, K., Sakata, J. and Eto, T. (1999). Marked increase of guanylin secretion in response to salt loading in the rat small intestine. Am. J. Physiol. 277, G960G966.
Macleod, K. M., Rodgers, R. L. and McNeill, J. H. (1981). Characterization of glucagon-induced changes in rate, contractility and cyclic AMP levels in isolated cardiac preparations of the rat and guinea pig. J. Pharmacol. Exp. Ther. 217, 798804.[Medline]
Mery, P. F., Brechler, V., Pavoine, C., Pecker, F. and Fischmeister, R. (1990). Glucagon stimulates the cardiac Ca2+ current by activation of adenylyl cyclase and inhibition of phosphodiesterase. Nature 345, 158161.[Medline]
Nakazato, M. Yamguchi, H., Date, Y., Miyazato, M., Kangawa, K., Goy, M. F., Chino, N. and Matsukura, S. (1998). Tissue distribution, cellular source, and structural analysis of rat immunoreactive uroguanylin. Endocrinol. 139, 52475254.
Parmley, W. W., Glick, G. and Sonnenblick, E. H. (1968). Cardiovascular effects of glucagon in man. N. Engl. J. Med. 279, 1217.[Medline]
Pavoine, C., Brechler, V., Kervran, A., Blache, P., Le-Nguyen, D., Laurent, S., Bataille, D. and Pecker, F. (1991). Miniglucagon [glucagon-(1929)] is a component of the positive inotropic effect of glucagon. Am. J. Physiol. 260, C993C999.
Plisetskaya, E. M. and Mommsen, T. P. (1996). Glucagon and glucagon-like peptides in fishes. Int. Rev. Cytol. 168, 187257.[Medline]
Pollock, H. G., Hamilton, J. W., Rouse, J. B., Ebner, K. E. and Rawitch, A. B. (1988a). Isolation of peptide hormones from the pancreas of the bullfrog (Rana catesbeiana). J. Biol. Chem. 263, 97469751.
Pollock, H. G., Kimmel, J. R., Ebner, K. E., Hamilton, J. W., Rouse, J. B., Lance, J. B. and Rawitch, A. B. (1988b). Isolation of alligator gar (Lepisosteus spatula) glucagon, oxyntomodulin, and glucagon-like peptide: amino acid sequences of oxyntomodulin and glucagon-like peptide. Gen. Comp. Endocr. 69, 133140.[Medline]
Prasad, K. (1975). Glucagon-induced changes in the action potential, contraction, and Na+-K+-ATPase of cardiac muscle. Cardiovasc. Res. 9, 355365.[Medline]
Raufman, J.-P., Singh, L., Singh, G. and Eng, J. (1992). Truncated glucagon-like peptide-1 interacts with exendin receptors on dispersed acini from guinea pig pancreas. J. Biol. Chem. 267, 2143221437.
Rombout, T. H. W. M., Van Der Grinten, C. P. M., Peeze Binkhorst, F. M., Taberne-Thiele, J. J. and Schooneveld, H. (1986). Immuno-cytochemical identification and localization of peptide hormones in the gastro-entero-pancreatic (GEP) endocrine system of the mouse and a stomachless fish, Barbus conchonius. Histochem. 84, 471483.
Santer, R. M. (1985). Morphology and innervation of the fish heart. Adv. Anat. Embryol. Cell Biol. 89, 1102.[Medline]
Smitherman, T. C., Osborn, R. C. and Atkins, J. M. (1978). Cardiac dose-response relationship for intravenously infused glucagon in normal intact dogs and man. Am. Heart J. 96, 363371.[Medline]
Uesaka, T. (1996). Synergistic action of neuropeptide Y and adrenaline in the eel atrium. J. Exp. Biol. 199, 18731880.
Uesaka, T., Yano, K., Sugimoto, S. and Ando, M. (1996). Effects of eel neuropeptide Y on ion transport across the seawater eel intestine. Zool. Sci. 13, 341346.[Medline]
Uesaka, T., Yano, K. Yamasaki, M. and Ando, M. (1994a). Glutamate substitution for glutamine at position 5 or 6 reduces somatostatin action in the eel intestine. Zool. Sci. 11, 491494.[Medline]
Uesaka, T., Yano, K., Yamasaki, M., Nagashima, K. and Ando, M. (1994b). Somatostatin-related peptide isolated from the eel gut: effects on ion and water absorption across the intestine of the seawater eel. J. Exp. Biol. 188, 205216.
Uesaka, T., Yano, K., Yamasaki, M. and Ando, M. (1995). Somatostatin-, vasoactive intestinal peptide-, and granulin-like peptides isolated from intestinal extract of goldfish, Carassius auratus. Gen. Comp. Endocrinol. 99, 298306.[Medline]
Yasuda, M., Uesaka, T., Furukawa, Y. And Ando, M. (1996). Regulation of atrial contraction in the seawater-adapted eel, Anguilla japonica. Comp. Biochem. Physiol. 113A, 165172.