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Glucagon-like peptide isolated from the eel intestine: effects on atrial beating

Toshihiro Uesaka^1,3, Keiichi Yano², Seiji Sugimoto² and Masaaki Ando^1,*

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

View larger version (44K): [in a new window]   Fig. 1. Final HPLC purification of EI-14 obtained from eel gut. (A) The active fraction obtained by chromatographic steps was subjected to reverse-phase HPLC (TSK ODS-80TM column) and eluted isocratically with 26.5% acetonitrile in 5% 2-propanol and 0.1% trifluoroacetic acid (pH 2.2). The flow rate was 0.5mlmin-1. (B) Effects of purified EI-14 on atrial beating and contractile force.

View larger version (16K): [in a new window]   Fig. 2. A comparison of the chemical characteristics of native (N) and synthetic (S) EI-14. (A) Reverse-phase chromatograms of N, S and N+S using the TSK ODS-120T column. N, S and N+S were eluted with a 50min linear gradient of 20%–30% acetonitrile in 10% 2-propanol and 0.1% trifluoroacetic acid. The flow rate was 0.5mlmin-1. (B) Cation-exchange chromatograms. Each peptide was eluted with a 25min linear gradient of 0-0.5moll-1 NaCl in 10% ethanol and 20mmoll-1 phosphate buffer (pH 6.7). The flow rate was 0.5mlmin-1.

View larger version (16K): [in a new window]   Fig. 3. Concentration–response curve for the effects of synthetic EI-14 on atrial beating. (A) The change in contractile force after addition of EI-14 plotted against its corresponding concentration (logarithmic scale). The control value before addition of EI-14 was 14.34±1.95mN (N=13). Values are means ± S.E.M. The number of experiments at each concentration is indicated in parentheses. (B) The effect of EI-14 on atrial beating rate. The control value was 69.5±6.6beatsmin-1 (N=13). The sample size is the same as in A.

View larger version (24K): [in a new window]   Fig. 4. Oscillation of intracellular Ca2+ concentration ([Ca2+]i) in a region of the atrial myocardium. The [Ca2+]i was determined by measuring the brightness of all pixels (N=1200) in the region, and the mean brightness ± S.E.M. was obtained. Error bars were all smaller than the size of the symbol. EI-14 (10-7moll-1) was applied at time zero.

View larger version (44K): [in a new window]   Fig. 5. Effects of extracellular Ca2+ on atrial beating. (A) Representative control response after treatment with synthetic EI-14 in normal Ringer’s solution. EI-14 (10-7moll-1) was applied during the period indicated by the horizontal bar. (B) Effects of EI-14 in the absence of Ca2+ in the bathing medium. Ca2+ was omitted during the period indicated by the horizontal bar (Ca2+-free). (C) Effects of EI-14 after pretreatment with verapamil. Verapamil (10-5moll-1) was applied during the period indicated by the horizontal bar.