PURIFICATION, CHARACTERIZATION, AND SPASMOGENIC ACTIVITY OF NEUROTENSIN FROM THE TOAD BUFO-MARINUS

Neurotensin (NT) was isolated from an extract of the intestine of the cane toad, Bufo marinus and its primary structure established as: u-Al a-Ile-Val-Ser-Lys-Ala-Arg-Arg-Pro-Tyr-Ile-Leu. This amino acid sequenc e shows five substitutions (Leu(2) --> Ala, Tyr(3) --> Ile, Glu(4) --> Val, Asn(5) --> Ser, and Pro(7) --> Ala) compared with bovine NT. Syn thetic Bufo NT (pD(2) = 8.05 +/- 0.28) was equipotent and equally effe ctive as bovine NT (pD(2) = 8.24 +/- 0.38) in producing spasmogenic co ntraction of isolated segments of toad small intestine. However, the m aximum response produced by Bufo NT was only 35 +/- 2% of that produce d by substance P. The potencies, but not the maximum responses, to Buf o and bovine NT were significantly (p < 0.05) attenuated by pre-treatm ent with atropine but neither parameter was significantly diminished b y tetrodotoxin and indomethacin. The data suggest that the action of N T involves interaction with receptors on toad intestinal smooth muscle that recognize the C-terminal region of NT (residues 8-13) that has b een fully conserved during evolution of tetrapods. Contractile activit y is mediated, at least in part, by release of acetylcholine. (C) 1998 Elsevier Science Inc.