Pharmacological and biochemical investigation of receptors for the toad gut tachykinin peptide, bufokinin, in its species of origin

Abstract

This is the first report of the development of a new radioligand [125I]Bolton-Hunter bufokinin ([125I]BH-bufokinin) and its use in the characterisation of tachykinin receptors in the small intestine of the cane toad, Bufo matrinus. The binding of [125I]BH-bufokinin to toad intestinal membranes was rapid, saturable, of high affinity and to a single population of binding sites with KD 0.57 nM and Bmax 3.1 fmol mg wet weight tissue(-1). The rank order of affinity of tachykinins to compete for [125I]-BH bufokinin binding revealed similarities with that of the mammalian NK1 receptor, being bufokinin (IC50, 1.7 nM)>physalaemin (6.7 nM)>substance P (SP, 10.7 nM)> or =neuropeptide gamma (NPgamma, 12.4 nM)> or =kassinin (17.8 nM)>scyliorhinin I (35.3 nM)> or =eledoisin (40.6 nM)> or =carassin (43.2 nM)> or =neurokinin A (NKA, 57.8 nM)> or =neurokinin B (NKB, 77.5 nM)>scyliorhinin II (338 nM). The mammalian NK3-selective agonist senktide was a very weak competitor. The radioligand [125I]neurokinin A showed no specific binding to toad intestinal membranes. In the toad isolated small intestine, the maximum contractile response to bufokinin was over 150% greater than that to acetylcholine in longitudinal muscle, whereas responses to bufokinin and acetylcholine were similar in circular muscle. Bufokinin was the most potent agonist (EC501 0.34 nM) and produced a long-lasting contraction. Other tachykinins such as physalaemin, SP and kassinin were also potent contractile agents. The potency values of mammalian and amphibian tachykinins derived from functional studies (pD2) correlated significantly with those from binding assays (pKi). The data for fish and molluscan tachykinins, however, showed poor correlation. Contractions to bufokinin and SP were unaffected by atropine, indomethacin and tetrodotoxin. The highly selective NK1 receptor antagonists CP 99994, GR 82334 and RP 67580 were ineffective in both binding and functional studies. Bufokinin increased inositol monophosphate formation in a concentration-dependent manner with an EC50 value of 10.7 nM, suggesting that the tachykinin receptor may be coupled to phosphoinositol hydrolysis. In summary, this study provides evidence for a high-affinity, bufokinin-preferring, NK1-like tachykinin receptor in the toad small intestine. This is probably not the receptor which mediates contraction to carassin, scyliorhinin II and eledoisin. The study also provides evidence that bufokinin and its receptor play an important physiological role in regulating intestinal motility.